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GUDMAP Protocols provides experimental protocols and standard specifications by a variety of groups from the GenitoUrinary Development Molecular Anatomy Project (GUDMAP). GUDMAP upgraded their website infrastructure and has integrated their resources with the (Re)BuildingaKidney consortium. For most current data, please visit GUDMAP: protocols.
(last updated: Dec 9, 2021)
Information Protocols| Title | Category | Equipment | Reagent/Solution | Procedure | Timing | Author | |
|---|---|---|---|---|---|---|---|
| Immunohistochemistry (IHC) for In-Situ Hybridization (ISH)-Stained Mouse Sections | Immunohistochemistry | avidin blocking solution (Vector Labs# SP-2001), biotin blocking solution (Vector Labs# SP-2001), Mouse IgG Blocking Reagent (Vector Labs #MKB-2213), bovine serum albumin (Fisher #BP1600-100), Blocking Reagent (Roche Applied Science # 11096176001), biotinylated goat anti-mouse, goat anti-rabbit, or rabbit anti-goat secondary antibodies (Vector Lab # BA-9200, BA-1000, BA-5000), avidin biotin complex (ABC) reagent (Vector Labs # PK6100), 3, 3'-diaminobenzidine (DAB) solution (Vector Labs # SK-4100) | Day 1: 1. Remove the 4% PFA and wash sections for 2 X 5 min at 25°C in TBS containing 0.1% TritonX-100 (TBSTx). 2. Incubate sections for 5 min at 25°C in 3.0% H2O2 in 1X TBSTx. 3. Incubate sections for 25 min at 25°C in 0.3% H2O2 in 1X TBSTx. 4. Wash sections 4 X 5 min at 25°C in TBSTx. 5. Incubate sections for 20 min at 25°C in avidin blocking solution (Vector Labs# SP-2001). 6. Rinse sections briefly with TBSTx. 7. Incubate sections for 20 min at 25°C in biotin blocking solution (Vector Labs# SP-2001). 8. Wash sections 2 X 4 min at 25°C in TBSTx. 9. For IHC with primary antibodies made in mouse species (for non-mouse primary antibodies, skip to step #10): A. Incubate sections for 1 hr at 25°C in a working solution of M.O.M. Mouse IgG Blocking Reagent (Vector Labs #MKB-2213). B. Wash sections for 2 X 3 min at 25°C in TBSTx. C. Proceed to step #11. 10. For IHC with primary antibodies made in non-mouse species: Incubate sections for 1 hr at 25°C in TBSTx containing 1% bovine serum albumin (Fisher #BP1600-100), 5% heat-inactivated serum from host-animal species of secondary antibody, and 1% Blocking Reagent (Roche Applied Science # 11096176001, diluted from a 10% stock solution of Blocking Reagent dissolved in maleic acid buffer as directed by the manufacturer). This Blocking Buffer is referred to as RBTx buffer. 11. Incubate sections overnight at 4°C in RBTx buffer containing primary antibody (concentration determined empirically). DAY 2: 12. Remove primary antibody and wash sections for 6 x 5 min at 25°C in TBSTX. 13. Incubate sections for 1 hr at 25°C in RBTx containing biotinylated goat anti-mouse, goat anti-rabbit, or rabbit anti-goat secondary antibodies (Vector Lab # BA-9200, BA-1000, BA-5000, concentration determined empirically). 14. Towards the end of step #13, dilute avidin biotin complex (ABC) reagent (Vector Labs # PK6100) in TBSTx according to the manufacturer’s instructions and incubate for at least 30 min at 25°C. 15. Wash sections for 6 x 5 min at 25°C in TBSTx. 16. Incubate sections 45 min at 25°C in diluted ABC reagent. 17. Wash 6 x 5 min at 25°C in TBSTx. 18. Prepare 3, 3'-diaminobenzidine (DAB) solution (Vector Labs # SK-4100) in TBSTx as described by the manufacturer. 19. Remove sections from tube and place in petridish. Remove TBSTx and add 1X DAB solution onto sections and monitor under microscope for color development (appr. 1-10 min). 20. As soon as color becomes visible, dilute the DAB with excess TBSTw and post-fix in 4% PFA. | Vezina Group | |||
| In situ hybridisation on vibrating microtome-cut mouse tissue sections | In situ hybridization | Day 1 PBSTw: 1X PBS containing 0.1% Tween-20 and 0.2mM sodium azide. Pass through 0.2µm filter to remove insoluble material/contaminants. 6% H2O2: 1mL 30% H2O2 per 4mL PBS Proteinase K: 0.25µL 20mg/mL proteinase K per 1mL PBSTw Post-fix: 8µL 25% glutaraldehyde per 1mL 4% PFA Prehybridization solution 100% formamide 50% 500mL 20X SSC 5X 250mL Blocking reagent 1% 10g 10mg/mL yeast tRNA 10µg/mL 1mL 10mg/mL heparin 10µg/mL 1mL dH2O to vol. -- to 1000mL Solution 1 100% formamide 50% 250mL 20X SSC 5X 125mL dH2O -- 75mL 10% SDS 1% 50mL Day 2 Solution 2 1M Tris-HCl, pH 7.5 10mM 5mL 5M NaCl 0.5M 50mL 100% Tween-20 0.1% 0.5mL 0.2M sodium azide 0.2mM 0.5mL dH2O -- 444mL Solution 3 20X SSC 2X 50mL 100% formamide 50% 250mL dH2O -- 200mL Tissue blocking (TB) buffer 10X TBS 1X 50mL 100% sheep serum 10% 50mL 10% blocking reagent 1% 50mL BSA 1% 0.5g dH2O to vol. -- to 500mL 100% Tween-20 0.1% 0.5mL 0.1% 0.5mL Antibody dilution (AD) buffer 10X TBS 1X 50mL 100% sheep serum 5% 25mL 10% blocking reagent 1% 50mL BSA 1% 0.5g dH2O to vol. -- to 500mL 100% Tween-20 0.1% 0.5mL Mix TBS, serum, blocking reagent, BSA & dH2O as shown above. Antibody absorption (AA) buffer 1X TBSTw -- 17mL 100% sheep serum 5% 1mL 10% blocking reagent 1% 2mL BSA 1% 0.2g embryo powder -- 0.12g 10% Blocking reagent maleic acid 100mM 1.2g 5M NaCl 150mM 3mL dH2O to vol. -- to 100mL Blocking reagent 10% 10g Day 3 2M Levamisole: Dissolve 4.82g levamisole in ~7mL double-distilled H2O (total volume should equal 10mL), aliquot 200µL volumes & store stocks at –20°C. TBSTw + levamisole: 1X TBSTw containing 2mM levamisole. NTMT + levamisole (inhibits endogenous alkaline phosphatases): 1M Tris-HCl, pH 9.5 100mM 50 mL 5M NaCl 100mM 10mL 1M MgCl2 50mM 25mL dH2O -- 415mL 100% Tween-20 0.1% 0.5 mL 2M Levamisole 2mM 0.5mL 3% H2O2: 1mL 30% H2O2 per 9mL PBSTw. Reagents and Supplies Anti-DIG antibody, Fab fragments, cat # 11214667001, Roche Blocking reagent, cat # 11096176001, Roche BM Purple AP substrate, precipitating, cat # 11442074001, Roche BSA, cat # BP1600-100, Fisher Formamide, cat # F5786-1L, Sigma Glutaraldehyde, 25% solution in H2O, cat # G6257-100ML, Sigma Heparin, sodium salt, cat # H3393, Sigma Hydrogen peroxide, 30% solution in H2O, cat # BP2633-500, Fisher Levamisole, cat # L9756, Sigma Magnesium chloride, cat # M33-500, Fisher Maleic acid, cat # M0375-500G, Sigma Paraformaldehyde, cat # 101176-014, VWR PBS, w/out Ca & Mg, MP Biomedicals powdered media, cat # ICN1760420, Fisher Proteinase K solution, 20mg/mL, biotechnology grade, cat # E195-5ML, Amresco RNase, cat # R6513, Sigma SDS, cat # S529-500, Fisher Sheep serum, cat # S2263-500mL, Sigma Sodium azide, granular, cat # S227I-100, Fisher Sodium chloride, cat # BP358-212, Fisher SSC, 20X solution, cat # S24022-4000.0, Research Products International Tris-HCl, cat # BP153-1, Fisher Tween-20, cat # BP337-100, Fisher Yeast tRNA, cat # 109495, Roche Polyester mesh, 33 micron, 12” x 24”, cat # CMY-0033-D, Small Parts Inc. | DAY 1 – All incubation steps to be performed with gentile agitation on an orbital shaker, unless otherwise indicated. 1. Prep baskets A. Close basket cap, affix sticker to cap and label with probe name. B. Use heated 18 gauge needle to puncture two holes per basket cap (facilitates air movement). 2. Prepare supplies and preheat solutions. A. Transfer prehyb stock solution into sealed conical tube and preheat to 60.5°C. B. Fill a small plastic storage container (used as a hybridization chamber) with about ½ inch of tap water, cover container and preheat to 60.5°C. 3. Prep samples. A. Use spring scissors to remove most of the agarose from the vibrating microtome-cut tissue sections. B. Remove any debris if stuck to tissue sections. C. Add 2mL PBSTw and one basket to each well of a 24-well culture plate. D. Transfer sections into labeled baskets. 4. Incubate tissues for 30 min at 25°C in 2mL/well of 6% H2O2. 5. Wash tissues 4 x 5 min at 25°C with 2mL/well PBSTw. 6. Incubate tissues 12 min at 25°C in 2mL/well proteinase K solution. 7. Wash tissues 1 x 5 min at 25°C with 2mL/well PBSTw. 8. Incubate tissues 20 min at 25°C in 2mL/well post-fix solution. 9. Wash tissues 2 x 5 min at 25°C with 2mL/well PBSTw. 10. Prehybridization step: Incubate tissues inside hybridization chamber for at least 1 hr at 60.5°C in 2mL/well prehyb buffer. 11. Hybridization step: Add 0.65mg probe/well and incubate sections overnight in hybridization chamber at 60.5°C in probe+prehyb buffer. 12. Prepare and preheat solutions for Day 2: Add SDS to solution 1 (to a final concentration of 1%) and preheat overnight at 60.5°C. DAY 2 - All incubation steps to be performed with gentile agitation on an orbital shaker, unless otherwise indicated. 1. Tissue sections are to remain in the baskets. Remove hyb buffer from each well and wash tissues 3 X 30 min at 60.5°C with 2mL/well Solution 1. 2. Prepare and preheat solutions. A. Prepare 50/50% mix of Solution1/Solution 2 and preheat to 60.5°C. B. Add RNase to Solution 2 and preheat to 37°C. C. Preheat one wash volume of Solution 3 to 25°C and two wash volumes to 60.5°C. 3. Wash tissues 1 X 10 min at 60.5°C with 2mL/well of 50/50% mixture of Solution 1/ Solution 2. 4. Wash tissues 4 X 10 min at 25°C with 2mL/well Solution 2. 5. Incubate tissues 15 min at 37°C in 2mL/well RNase solution. 6. Wash tissues 1 X 10 min at 25°C with 2mL/well Solution 2. 7. Wash tissues 1 X 10 min at 25°C with 2mL/well Solution 3. 8. Wash tissues 2 X 1 hr at 60.5°C with 2mL/well Solution 3. 9. Preheat Tissue Blocking (TB), Antibody Dilution (AD), and Antibody Absorption buffers to 25°C. 10. Wash tissues 3 X 10 min at 25°C with 2ml/well TBSTw. 11. Blocking step A. Incubate tissues at least 2 hr at 25°C in 2mL/well TB buffer. B. Add 3.3mL anti-DIG antibody per 600µL AA buffer & incubate at least 2 hr at 4°C. 12. Antibody step A. Centrifuge AA buffer containing antibody at 10,000 rpm for 1 min. B. Remove supernatant and add entire supernatant volume to 6mL AD buffer. C. In a humidified chamber, incubate tissues overnight at 4°C in 2mL AD buffer + antibody. Day 3 - All incubation steps to be performed with gentile agitation on an orbital shaker, unless otherwise indicated. 1. Remove antibody solution and save. A. Add sodium azide to a final concentration of 0.2mM to prevent microbial growth. B. Store antibody solution at 4°C and reuse up to two additional times. 2. Wash tissues 8 X 10 min at 25°C with 2mL/well TBSTw containing 2 mM levamisole. 3. Remove tissues from baskets, use forceps to separate sections & remove visible debris, transfer tissues into clean microcentrifuge tubes. 4. Wash tissues 1 X 10 min at 25°C with 1mL NTMT containing 2 mM levamisole. 5. Detection step A. Protect tissues from light and incubate at 25°C in 1mL/tube of a 60/40% mixture of NTMT containing 2mM levamisole/BM Purple. B. Color development time ranges from several hours to several days. Change NTMT/BM Purple solution as needed (substrate will precipitate over time). Once color is fully developed, wash tissues 2 X 5 min at 25°C with 1mL/tube NTMT containing 2mM levamisole. 6. Bleaching step A. Post-fix tissues overnight at 4°C in 1mL/tube 4% PFA. B. Remove PFA and incubate tissues for 30 min at 25°C in 1mL/tube PBSTw containing 3% H2O2. C. Wash tissues 1 X 10 min at 25°C in 1mL/tube PBSTw. D. Store tissues at 4°C in 1mL/tube 4% PFA. | DAY 1 – All incubation steps to be performed with gentile agitation on an orbital shaker, unless otherwise indicated. DAY 2 - All incubation steps to be performed with gentile agitation on an orbital shaker, unless otherwise indicated. Day 3 - All incubation steps to be performed with gentile agitation on an orbital shaker, unless otherwise indicated | Vezina Group | ||
| Riboprobe Synthesis | PCR | nylon membrane (Roche, cat# 1209272) | Roche DIG Nucleic Acid Detection kit - cat# 11175041910, Roche DIG Nucleic Acid Detection kit - cat# 11175041910, Roche DIG High Prime DNA Labeling and Detection starter kit protocol (cat# 11745832910), version December 2005, Maleic acid buffer (stable at 15-25°C) 0.1M Maleic acid (CAS# 110-16-7) 0.15M NaCl (CAS# 7647-14-5) Adjust pH to 7.5 w/ NaOH pellets (CAS# 1310-73-2) Washing buffer (stable at 15-25°C) 0.1M Maleic acid 0.15M NaCl Adjust pH to 7.5 w/ NaOH pellets Add 0.3% (v/v) Tween 20 (CAS# 9005-64-5) Detection buffer (stable at 15-25°C) 0.1M Tris-HCl 0.1M NaCl Adjust to pH 9.5 10x Blocking reagent (stable at 2-8°C or -15 to -25°C) ~50 g per bottle, comes with kit. Dissolve in Maleic acid buffer to a final concentration of 10% (w/v) = ~500ml. Requires heating and shaking (stir on heated magnetic stir plate ~20 min). Autoclave this stock solution. RNA dilution buffer (see p.3 of Roche DIG Labeling kit protocol – cat# 11175025910) DEPC H2O: 20x SSC: 37% Formaldehyde (CAS# 50-00-0) in the ratio 5:3:2 Blocking solution (120 ml per membrane – 100 ml for block, 20 ml for antibody solution) Prepare 1x working solution by diluting 10x blocking reagent 1:10 with Maleic acid buffer Antibody solution (20 ml per membrane) Centrifuge anti-digoxigenin-AP antibody 5 min at 10,000 rpm prior to use. Dilute antibody 1:5000 in blocking solution (4µl in 20ml). Color substrate solution (20 ml per membrane) Add 400 ml of NBT/BCIP stock solution to 20 ml detection buffer. Store protected from light. | 1. Primer Design Primers are designed to amplify a 500-1000 bp (750 bp ideal) region of the coding sequence of each gene. Primers are designed using Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3_www.cgi) then candidate primer sequences are analyzed using Net Primer (www.premierbiosoft.com/netprimer) to determine the most optimal pair. The reverse primer is linked to a SP6 polymerase promoter tag sequence as follows: Reverse (antisense) primer: 5’ – (leader sequence) GCG – (SP6 sequence) ATTTAGGTGACACTATAG – primer sequence - 3’ Primers are ordered from IDT (Integrated DNA Technologies) and resuspend in RNase-free water to make a 100µM stock. Make 20µM working dilutions for each primer, then combine the forward and reverse for each primer set. Example: 10µl of the 100µM Forward primer stock + 10µl of the 100µM Reverse primer stock + 30µl RNase-free water. Store stocks and working dilutions at -20°C. 2. PCR First round PCR: Set up 25µl reactions using Qiagen Taq PCR Master Mix Kit (cat # 201443) as follows: Final concentration Taq PCR master mix 12.5 µl2.5 U Taq, 200 µM dNTPs, 1x buffer, 1.5 µM MgCl2 20 mM F+R primer1 µl800 nM cDNA template * 11.5 µl *cDNA template is reverse transcribed from a pool of RNA isolated from fetal testes of various gestation ages. PCR Cycling conditions: 94°C 2 min1 cycle 94°C 20 sec 55°C 30 sec 35 cycles 72°C 1 min 72°C 10 min 1 cycle Electrophorese the entire 25µl reaction on a 1% agarose 1xTAE gel. Check for a single band of the right size, cut it out of the gel, and purify using Qiagen’s QiaQuick Gel Extraction kit (cat# 28706). Elute purified PCR products with 50µl elution buffer then set up a second PCR using the template generated in the first PCR. Second Round PCR: Set up 50µl reactions using Qiagen Taq PCR Master Mix Kit (cat # 201443) then run the same cycling conditions as the first round PCR: 1xFinal concentration Taq PCR master mix 25 µl 2.5 U Taq, 200µM dNTPs, 1x buffer, 1.5 µM MgCl2 20 mM F+R primer 2 µl 800 nM Template PCR product 2 µlH2O 21 µl Total rxn volume 50 µl Electrophorese the entire 50µl reaction on a 1% agarose 1xTAE gel. Check for single bands of the right size, cut them out of the gel, and purify using Qiagen’s QiaQuick Gel Extraction kit (cat# 28706). Elute purified PCR products with 50µl elution buffer. DNA concentration is measured using 2µl on a NanoDrop and another 2µl is electrophoresed on a 1% agarose 1xTAE gel to verify a single band of appropriate size. Sequence Verification: 100-200 ng of each PCR product is sent to MWG for sequence verification (ValueRead sequencing using SP6 reverse primer). Once sequences are confirmed, continue with production of DIG-labeled riboprobes. 3. In vitro Transcription of Digoxigenin-Labeled Riboprobes for in situ Hybridization Roche DIG RNA Labeling Kit (SP6/T7) – cat# 11175025910 Start with 200ng template PCR product, bringing volume to 13µl with RNase-free H2O. Keep samples and the following reagents on ice: 10x NTP labeling mix 2 µl 10x transcription buffer 2 µl Protector RNase Inhibitor 1 µl RNA polymerase SP6 (20U/ml) 2 µl Template + H2O 13 µl Total rxn volume 20 µl | Gaido Group | ||
| Riboprobe Synthesis for In Situ Hybridization | PCR | 10x Big Dye® v3.1 sequencing buffer 4.0 ABI* 10 µM T7 primer 0.5 Invitrogen Big Dye® v3.1 1.0 ABI 4337455 Sterile water 11.5 Purified plasmid (60+ ng/µl) 3.0 | Mix the agents at room temperature in a 0.2 ml tube; ensure homogeneity by mixing well and quickly spinning in a centrifuge • Add one drop of sterile, filtered mineral oil to each tube. • Use PCR machine that ramps temperature (do not use RoboCyclers). PCR conditions: 96o C 30 seconds 50o C 15 seconds 60o C 4 minutes 25 cycles • Use Edge BioSystems Performa spin columns (73328) for reaction purification. o Spin at 850g for 3 minutes to remove packing buffer. ƒ Do not let columns stand dry for more than five minutes. o Add reaction product (16-20 µl) to the center of gel column. Do not touch pipette tip to gel column or sidewall. o Spin at 850g for 3 minutes to elute purified product. o Transfer to 8-tube PCR strips. o Submit to Harvard’s MCB Sequencing Facility for analysis. Sequence Analysis • Analyze sequence with BLAT (http://genome.ucsc.edu/). • Use Entrez-Gene to research alternate gene names. • Use chromosome numbers to quickly identify suspected mismatches. • Three possible outcomes of research are match, no data, and mismatchBacteria Storage • Sequencing data MUST match supposed identity for permanent storage. • Re-pick original colony from master plate with a pipette tip. • Inoculate into 2 ml of 8% glycerol/LB/ampicillin in a 15 ml conical. • Grow overnight at 37°C with agitation. • Transfer to 2 ml screw-top vial (Sarstedt 72.694.006) and store at -80°C. Probe Template PCR Procedure generates DNA template for synthesis of riboprobe. We have experimented with various annealing temperatures; 46o C yields robust product with high specificity. • Use a 96-well PCR plate with cover. • Dilute purified plasmid to 1.0-5.0 ng/µL (typically 1:100). • Keep enzyme and plate on ice; mix the following: | McMahon Group | Phil Ingham Ron Conlon Barry Rosen and Richard Harland in David Wilkinson ed. In Situ Hybridization: A Practical Approach Oxford: IRL Press 1992. | ||
| RNA isolation from LCM samples | RNA extraction | Arcturus PicoPure RNA isolation kit (catalogue # KIT0204) | Follow manufacturer’s protocol for Arcturus PicoPure RNA isolation kit (catalogue # KIT0204). Note: The ‘optional’ DNase treatment is performed on all isolations. | Gaido Group | |||
| Digoxigenin-Labeled In Situ Hybridization for P1 Mouse Kidney Sections | image acquisition | Nikon DXM1200 digital camera attached to a Nikon Edge scope. | We photograph samples with a Nikon DXM1200 digital camera attached to a Nikon Edge scope. 2. 6 photos per gene: 1 global view at 100X and 5 regional (outter cortex, juxtamedullary cortex, outter medulla, inner medulla, renal pelvis), each at 4000X. White balance should be adjuted so that the color of the digital image matches the color of the real image. The center of the bladder generally provides a good white reference point. Non-expressing regions can also provide a white reference. 3. Naming protocol is “YYMMDDi[type][initial]_###”. For example, the eighth photograph of a whole mount in situ hybridization sample taken by John Harvard on May 15, 2001 is called “010515iWMjh_008.” Also, as each sample is photographed, a separate Excel document is maintained to record filename, gene name, MTF, zoom, and BM Purple development time | McMahon Group | |||
| Digoxigenin-Labeled In Situ Hybridization for E15.5 Wholemount Kidneys | n PBT (Boehringer 161519), 75% MeOH/0.85% NaCl solution. o 50% MeOH/0.85% NaCl solution. o 25% MeOH/0.85% NaCl solution. | Rehydrate samples in each of the following at room temperature for 5 minutes: o 75% MeOH/0.85% NaCl solution. o 50% MeOH/0.85% NaCl solution. o 25% MeOH/0.85% NaCl solution. o 2x PBT. Bleach in 6% hydrogen peroxide in PBT at room temperature for 30 minutes. o Wash 3x in PBT at room temperature for 5 minutes each. • Treat with 10 µg/ml Proteinase K in PBT (Boehringer 161519).??? o Timing is important. o Do not shake or rotate vial, but gently mix every 5 minutes. ? E14.5 UGS for 10 minutes. ? E15.5 UGS for 30 minutes. • “Quick” gentle wash in 2 mg/ml glycine/PBT (filtered). o Wash 2x in PBT at room temperature for 5 minutes each. • Fix in 0.2% glutaraldehyde/4% PFA in PBT at room temperature for 20 minutes. o Wash 2x in PBT at room temperature for 5 minutes each. • Place in pre-hyb solution and heat at 70°C for one hour. • Store in pre-hyb solution at -20°C for up to 1 month or hybridize immediately. Note: The 30 minute E15.5 Proteinase K time provides the best deep probe penetration. We tested 15, 20, 30, and 60 minute treatments with Wnt7b and verified with deep structure markers Hoxb7, Wnt7b, and Shh probes. These times did not affect superficial staining as shown by Wnt11, BF2, and Six2 probes. | McMahon Group | Phil Ingham Ron Conlon Barry Rosen and Richard Harland in David Wilkinson ed. In Situ Hybridization: A Practical Approach Oxford: IRL Press 1992. | |||
| Riboprobe Synthesis for In Situ Hybridization | sequencing analysis | • Analyze sequence with BLAT (http://genome.ucsc.edu/). • Use Entrez-Gene to research alternate gene names. • Use chromosome numbers to quickly identify suspected mismatches. • Three possible outcomes of research are match, no data, and mismatch | McMahon Group | ||||
| RNA amplification and labeling of LCM samples for microarrays | Aminoallyl-aRNA Amplification | Epicentre TargetAmp 2-Round Aminoallyl-aRNA Amplification kit 1.0 (catalogue # TAA2R4924) | Proposed kit and protocol to be followed is Epicentre TargetAmp 2-Round Aminoallyl-aRNA Amplification kit 1.0 (catalogue # TAA2R4924). Evaluation of this kit is still in progress. | Gaido Group | |||
| Whole-Mount Indirect Fluorescent Immunohistochemistry – Double or multiple staining | Fluorescent Immunocytochemistry | 4% paraformaldehyde in PBS (1:4, purchased 16%, diluted with PBS), blocking buffer(1% BSA, 0.1% Saponin, 0.02% sodium azide in PBS | Tissue samples are fixed with 4% paraformaldehyde in PBS (1:4, purchased 16%, diluted with PBS), overnight, 4 ºC. (Keep samples in tubes?) Rinse, 2 times in PBS. Quench in 50 mM NH4CL, 15 min. Block in blocking buffer, 24 hr (1% BSA, 0.1% Saponin, 0.02% sodium azide in PBS) [100mg BSA+10ul Saponin+2ul sodium azide ------à10 ml PBS]. You also need to try blocking with Triton X-100 (0.1%) instead of saponin. Incubated in FIRST primary antibody in fresh blocking buffer, 24 hr, 4 ºC. You can incubate all primary antibodies at the same time. Wash 5 times, 1 hr/each, in blocking buffer. Incubated in secondary antibody in fresh blocking buffer, 24 hr, 4 ºC. You can incubate all secondary antibodies at the same time. Wash 5 times, 1 hr/each, in blocking buffer. | Gaido Group | |||
| Fluorescent In Situ-Hybridization | In situ hybridization | solution and chemical list (http://www.gudmap.org/Research/Protocols/Gaido/GAIDO_FISH_3.html) | Starting the FISH run Flush the instrument. This can be done by pressing the “flush instrument” button in the menu bar of the Gemini program, and clicking “OK” when the description box comes up. The robot should flush about 50 ml of system liquid. Wipe the tips with 100% ethanol using a Kimwipe. Make sure the two water baths are on. Fill up the large system liquid water with ultra pure (Milli-Q) the night before a run, allow the liquid to degas naturally. Do not add system liquid the same day unless the water is degassed, you do not want bubbles in the system liquid which may cause a run to fail. Empty out the waste containers. Open Evoware, select the script you want to run. Press the start button and the script will begin and ask for the number of slides to be processed. Before entering the slides number and pressing enter, prepare the methanol peroxide solution and place into receptacle. Fluorescent In Situ-Hybridization Steps Prehybridization steps “Washing” means adding the amount of solution specified in the protocol to all flow-through chambers. “Repeated washing” means multiple cycles of washing. By contrast, “incubation” means that added solutions were left in the chambers for the amount of time specified in the protocol below. All solutions were provided in suitable containers located on the platform. Wash 5 times for 5 minutes with MeOH containing 0.7% hydrogen peroxide. Wash 7 times with 300µL of PBS. Incubate 2 times for 5 minutes of 0.2N HCl. Wash 7 times with PBS. Incubate 2 times for 15 minutes with pre-hybridization solution. Incubate 1 time for 15 minutes while the temperature is raised to 64°C (no solution is delivered, only a timer is set within the script, this gives the incubator time to warm before probe hybridization). Hybridization step The robot automatically delivers the probe to the specified hybridization chambers. Incubate probe at 64°C for 5.5 hours. After 2.5 hours a second aliquot of probe is pippetted. Adding fresh probe halfway through the incubation increases the signal strength of weakly expressed genes. Post hybridization stringency washes Post hybridization washes are carried out at 62°C. Temperature reduction to room temperature is carried out while in cycle 1 of step 1 below: Stringency wash solution is prewarmed for 1.5 hours (no longer) on the robot platform in a heated container so that they reach approximately 60°C. This is necessary to avoid degassing that would occur if room temperature solutions were added to a 62°C hybridization chamber. Incubate 5 times for 5 minutes with 5x SSC. Incubate 5 times for 10 minutes with 2x SSC with 50% formamide (Formamide I). Incubate 5 times for 12 minutes with 1x SSC with 50% formamide (Formamide II). Incubate 4 times for 8 minutes with 0.1x SSC. Probe detection reactions Temperature reduction to room temperature is carried out while in cycle 4 of step 4 in the above stringency washes. All post-hybridization steps are carried out at room temperature. Wash 4 times for 5 minutes with NTE Incubate 6 times for 5 minutes with 20mM iodoacetamide in NTE Wash 4 times for 5 minutes with NTE Wash 2 times for 5 minutes with TNT solution Incubate 6 times for 5 minutes with 4% sheep-serum Wash 4 times for 5 minutes with TNT solution Incubate 2 times for 10 minutes with TNB blocking buffer (PerkinElmer Life sciences, FP1020) Wash 2 times for 5 minutes with TNT solution Wash 2 times for 5 minutes with maleate wash buffer (MWB) Incubate 2 times for 10 minutes with 1% blocking reagent (Roche) Wash 2 times for 5 minutes with MWB Wash 2 times for 5 minutes with TNT solution Incubate 3 times for 5 minutes with TMN solution Wash 4 times for 5 minutes with TNT solution Incubate 4 times for 10 minutes with TNB blocking buffer. Incubate 2 times for 30 minutes with Anti-DIG-POD in TNB blocking buffer Incubate 6 times for 5 minutes with TNT solution Incubate 1 time for 30 minutes with tyramide-biotin diluted with amplification diluent’s buffer (all in TSA Plus Cy3 kit, PerkinElmer Life sciences NEL744). Wash 4 times for 5 minutes with TNT solution Wash 2 times for 5 minutes with TMN solution. Wash 3 times with system liquid water. Wash once with NTE. Incubate once for 10 minutes with 4% PFA Wash 3 times with system liquid water Disassembly of Hybridization chambers Remove hybridization chambers from the robotic platform. Use the clamp remover to remove clamps from assembled hybridization chambers. Place clamps into a small container to be rinsed. Place unclamped hybridization chambers face-down into MilliQ water one at a time. Do not allow chambers with slides to set in water for too long. Place slide frames into a container to be rinsed later. Carefully remove slides from water and back plates, let spacers float off and place them, in the metal slide racks. Take care not to scratch the tissue sections. Rinse the slide racks briefly in ultra-pure (Milli-Q) water. Allow the slides to dry in a fume hood at RT for ~10 minutes. Coverslip with VectaShield Mounting Medium with Dapi (Vector Laboratories, Inc. cat# H-1200). (use only 1-3 drops per slide, depending on the number of sections per slide) Cleaning of Hybridization chambers Take back plates from the water and place them into containers with ethanol. Leave them overnight. Take back plates out of ethanol and place them in stainless steel containers, and put them in a dishwasher to be cleaned. Rinse slide holders and clamps twice in deionized water and leave them out to dry. Remove stainless steel containers from dishwasher; place the containers in external stainless steel box and autoclave. Shut-down Empty out the waste containers. Fill up the system liquid container. Turn off the robot; water-baths can stay on stand-by. | DAY 1: Acetylation of frozen sections cut at 10µm Assembly of hybridization chambers Daily Solutions Starting the run Steps (Prehyb, hyb, post-hyb, probe detection reactions) DAY 2: Disassemble hyb chambers Clean-up Shut-down | Gaido Group | ||
| Microarrays | Microarray | Follow Affymetrix GeneChip Expression Analysis manual (part # 701021 rev. 4) for cRNA fragmentation, target hybridization, and array washing, staining and scanning. | Gaido Group | ||||
| Fluorescent Immunocytochemistry Protocol on Frozen sections | Fluorescent Immunocytochemistry | 4% paraformaldehyde (diluted 16% paraformaldehyde at 1:4 in PBS),Blocking buffer (500 ml normal goat serum to 10 ml PBS + 10 ml Triton, serum match to species the second antibody from), 3% BSA (300mg in 10 ml PBS) | Prepare cryostat section at 5-7 mm (for In situ, may 10 mm) Fix with 4% paraformaldehyde (diluted 16% paraformaldehyde at 1:4 in PBS), 10 min, room temperature Wash with PBS/Triton, 5 min X 3 Block with Blocking buffer (500 ml normal goat serum to 10 ml PBS + 10 ml Triton, serum match to species the second antibody from), 1 hr, room temperature Wash with PBS, 5 min X 1 Primary antibody diluted in 3% BSA (300mg in 10 ml PBS), overnight, at 4 °C Wash with PBS, 5 min X 3 Incubate with biotinylated secondary antibody, 1:500 dilution (pay attention to different species, against primary), 1 hr, room temperature Wash with PBS, 5 min X 3 Mount coverslips with VectaMount Visualization using microscope Analysis using Confocal Software | Gaido Group | |||
| Digoxigenin-Labeled In Situ Hybridization for E15.5 Wholemount Kidneys | wash | BioLane HTI | RNase A (Sigma R 6513), more solution details refer to http://www.gudmap.org/Research/Protocols/McMahon/hyb_solution_guide.pdf | Place BioLane intake tubes in a large flask of clean water; run cleaning program. • Load samples with plastic transfer pipette into nylon-mesh BioLane plate in a tray containing warm Solution I. • Load tray into BioLane System 1 or System 2. • Place intake tubes into solutions. Consult accompanying BioLane guide. • Place output tube into a hazardous waste jug (formamide waste). • Do not have MBST tube in place for first cycle (tray is already full). • Start appropriate program; wait for first intake cycle; replace MBST tube. • Remove hazardous waste jug after final Solution III wash (approximately 4 hours from start time). • The HTI machine uses 40 ml solution per wash in a half tray; 75 ml solution per wash in a full tray; 150 ml solution per wash in a double tray; and 300 ml solution per wash in a double system, double tray procedure. Overview of Wash Procedure • Wash 4x with Solution I at 70°C for 15 minutes each. • Wash once with 1:1 mixture of Solution I to Solution II ?at 70°C for 10 minutes. • Wash 3x with Solution II at room temperature for 5 minutes each. • Wash with 100 µg/ml RNase A (Sigma R 6513) in Solution II at 37°C for one hour. • Wash once with Solution II at room temperature for 5 minutes. • Wash once with Solution III at room temperature for 5 minutes. • Wash 4x with Solution III at 65°C for 15 minutes each. • Wash 3x with MBST at room temperature for 5 minutes each. • Wash in 10% HISS blocking solution at room temperature for 4 hours. • Incubate in anti-digoxigenin alkaline phosphatase 1:5000 diluent (Roche 1093274) in 1% HISS blocking solution at 4°C for 12 hours. • Wash 3x with MBST at room temperature for 5 minutes each. Wash 10x with MBST at room temperature for one hour each. • Wash 8x with MBST at 4°C for 2-5 hours each. • Samples can be held in MBST at 4°C for up to 2 days. • Wash 3x in NTMT at room temperature for 5 minutes each | McMahon Group | Phil Ingham Ron Conlon Barry Rosen and Richard Harland in David Wilkinson ed. In Situ Hybridization: A Practical Approach Oxford: IRL Press 1992. | |
| Semi-Automated Whole Mount in situ Hybridisation | In situ hybridization | BioLane HTI Robot | DAY1 Methanol 70mL PBTX 350mL PBT 80mL PBT/H2O2 50mL Proteinase K 50mL Glut/PFA/PBTX 40mL PBT/H2O2 10mL H2O2 40mL PBT Proteinase K (Roche3115879) 25uL 20mg/mL stock proteinase K 50mL PBTX Glut/PFA/PBTX 320uL 25% glutaraldehyde 48uL Triton X-100 40mL 4% PFA in PBS DAY2 Pre-Block 50mL Antibody 50mL Solution1 70mL 2xSSC 70mL 2xSSC+CHAPS 80mL 0.2XSSC+CHAPS 80mL TBTX 180mL TBTX+0.1%BSA 200mL NTMT 120mL Pre-Block (100mL) N.B. If re-using antibody only require 50mL 2g BSA 10mL Sheep Serum 90mL TBTX Antibody – Can store in fridge and re-use up to 3 times 15uL anti-DIG antibody (Roche11093274910) 3mL Pre-Block 18mg Embryo powder Incubate this solution on rocker in cold room for 3 hours minimum and then spin down at 13 000 rpm for 10min. Collect the supernatant and add to 50mL pre-block solution before using in the in situ hybridisation. Solution 1 50mL Formamide 25mL 20x SSC 100uL Triton X-100 10mL 5% CHAPS 15mL water 2xSSC+CHAPS (80mL) 2xSSC+CHAPS (100mL) 8mL 20xSSC 10mL 20xSSC 1.6mL 5% CHAPS 2mL 5% CHAPS 70.4mL H2O 88mL H2O 0.2xSSC+CHAPS (80mL) 0.2xSSC+CHAPS (100mL) 0.8mL 20x SSC 1mL 20x SSC 1.6mL 5% CHAPS 2mL 5% CHAPS 77.6mL H2O 97mL H2O TBTX+0.1%BSA 0.2g BSA 200mL TBTX NTMT 12mL 1M NaCl 12mL 1M Tris.Cl pH9.5 6mL 1M MgCl2 120uL Tween 20 90mL H2O | Pretreatment of Embryos 1. Dissect embryos of 9.5dpc (whole embryo), 10.5dpc (forelimb-hindlimb) and 12.5dpc(urogenital tract) in ice-cold PBS. 2. Immediately fix the tissues with 4% paraformaldehyde (PFA) overnight (16-18hours) at 4°C. 3. Wash the tissues twice with PBTX for 10 minutes each at room temperature. 4. Dehydrate with a series of 25% MeOH/75%PBTX, 50%MeOH/50%PBTX, 75% MeOH/25%PBTX washes for 20 minutes each, follow by two washes of 100% MeOH for 20minutes. 5. Store embryo tissues in 100%MeOH at -20°C. Day 1- Cleaning, Re-hydration and Hybridisation (Blue System) 1. Place tray with nylon mesh baskets into the blue system and run the cleaning program. 2. Wash all the tubing in the blue system with 0.2M NaOH. (This is to maintain an RNase-free environment) 3. Wash all the tubing in the blue system with RO-H2O. 4. Carefully put the embryonic tissues into the baskets. Each basket contains 2x 9.5dpc, 3x 10.5dpc, 1 male and 1 female 12.5dpc, and 2 x12.5dpc/2d explants. 5. Start the re-hydration program of the blue system. 6. The samples are treated as follows: Re-hydration RT 5 Minutes100% MeOH RT 5 Minutes75% MeOH/ 25% PBTX RT 5 Minutes 50% MeOH/ 50% PBTX RT 5 Minutes25% MeOH/ 75% PBTX RT10 Minutes PBTX RT 5 MinutesPBT RT60 Minutes PBT/ 6% H2O2 RT 5 Minutes PBT RT 5 Minutes PBTX RT 5 Minutes PBTX Digestion RT 20 Minutes 10ug/ml Proteinase K/ PBTX RT 5 MinutesPBTX RT 5 MinutesPBTX 7. Turn off robot and wash samples with 0.2% gluteraldehyde/4% PFA in PBTX in the fume hood at room temperature for 20 minutes. 8. Wash twice with PBTX at room temperature for 10 minutes each. 9. Remove baskets from tray and place baskets into a 48 well Cellstar plate containing 0.5ml pre-hybridisation solution in each well at 65°C for 2 hours. 10. Transfer baskets to a 48 well Cellstar plate with 0.5ml hybridisation solution at 65°C overnight. (Hybridisation solution = pre-hybridisation solution + ~0.2µg/ml DIG-labelled RNA probe.) Preparation 11. Prepare the post-hybridisation washes and place at 65°C overnight. Preabsorption of anti-DIG antibody 12. Prior to starting post-hybridisation washing, prepare the pre-blocking solution and pre-absorb the anti-DIG antibody Roche11093274910. (The pre-absorbed anti-DIG antibody can be kept at 4°C and recycled up to 3 times.) 18mg of embryo powder is placed in a 10ml tube with 10% sheep serum, 2% BSA in TBTX and 15µl anti-DIG antibody. Incubate at 4°C for 3 hours or longer with gentle rocking. Spin sample in a microfuge for 10 minutes, 4°C at 13000rpm. Collect the supernatant and dilute it with 10% sheep serum, 2% BSA in TBTX. Store the antibody at 4°C until use. DAY 2 – Post-Hybridisation and pre-blocking (Red system) 1. Remove the baskets from the 48 well plate and place in a tray filled with solution 1. 2. The robot will preheat the rocker with the samples to 65°C for 15 minutes. 3. The following stringency washes are performed: Post-Hybridisation 65°C 5 Minutes100% Solution 1 washes 65°C 5 Minutes 75% Solution1/25% 2x SSC 65°C 5 Minutes 50% Solution 1/50% 2x SSC 65°C 5 Minutes 25% Solution 1/75% 2x SSC 65°C10 Minutes 2x SSC / 0.1% CHAPS 65°C 10 Minutes 2x SSC / 0.1% CHAPS 65°C 5 Minutes 0.2x SSC / 0.1% CHAPS 65°C 5 Minutes 0.2x SSC / 0.1% CHAPS RT 10 Minutes TBTX RT 10 Minutes TBTX Pre-blocking RT 2 Hours Pre-Block Solution 4°C Overnight Pre-absorb anti-DIG antibody Preparation 4. Prepare 0.1% BSA / TBTX at the end of day 2 and insert tubing into solution. The robot will start taking in 0.1% BSA / TBTX early morning of day 3. Day 3 – Post-antibody washes and Development 1. The samples are washed as follows: Post-Antibody RT30 Minutes0.1% BSA / TBTX washes RT30 Minutes0.1% BSA / TBTX washes RT30 Minutes0.1% BSA / TBTX washes RT30 Minutes0.1% BSA / TBTX washes RT30 Minutes0.1% BSA / TBTX washes RT10 MinutesTBTX The pH for NTMT decreases over time so it needs to be made fresh. The robot will pause for you to make up fresh NTMT and re-start it washes RT10 MinutesTBTX washes RT10 MinutesNTMT washes RT10 MinutesNTMT washes RT10 MinutesNTMT 2. Transfer contents of the baskets into 12 well plates with 1ml of BM purple substrate Roche11442074001 in each well. Bring the BM purple AP substrate to 15°C –20°C before using, no dilution is required but you may need to filter the solution to remove any precipitate. 3. Wrap the 12 well plates with aluminium foil and monitor samples for colour development approximately every 15-30 minutes. Note: Normally 3 control samples will be used to verify the success of the run. Controls are Wnt4 (strong), Wnt7b (medium) and Shh (weak). For Wnt4 and Wnt7b, generally the expression develops within 1 to1.5hours whereas Shh can take 25-30 hours. 4. When the colour has proceeded to the desired extent, stop the colour development by transferring the samples to a new 12 well plate containing distilled water. Depending on the extent of colour development, samples can be left overnight to continue developing or kept at 4°C to slow down the reaction. Note: If the samples have been over-developed, wash several times in PBS with 1% Triton X-100. This can be done for several days if necessary and will decrease the background issue. 4. Wash 3x with PBS at room temperature for 5 minutes each. 5. Fix samples with cold 4% paraformaldehyde at room temperature for 30 minutes. 6. Wash 3x with PBS at room temperature for 5 minutes each. 7. Store samples in 2ml Eppendorf tubes with PBS at 4°C. 8. Photograph as soon as possible. Use a petri dish with 1% agarose as a background. | Day 1- Cleaning, Re-hydration and Hybridisation (Blue System), DAY 2 – Post-Hybridisation and pre-blocking (Red system), Day 3 – Post-antibody washes and Development | Little Group | PMID:18618131 |
| Optimized Vibratome Section in situ Hybridization for P1 Mouse Kidney Sections | fixation | 1. Dissect newborn kidneys in cold PBS. 2. Fix kidneys in 4% paraformaldehyde for 1hr. 3. Blot dry kidneys on paper towel briefly (Important! Otherwise kidneys are easy to pop out of the section during sectioning). 4. Embed kidneys in 15%gelatin (300 Bloom)/PBS in cryomolds and set at 4C for 1 hour. Note: Gelatin of 175 and 300 Bloom were tested. 300 Bloom gelatin makes blocks of higher hardness which is important for sectioning of consistent thickness. 5. Take gelatin blocks out of cryomolds, fix blocks with 4%PFA for 18 hrs at 4C. Note: Fixations of 6h and 18h were tested with probes of cortex and medulla, stroma and epithelium (BF2 and Wnt7b probes). 18h fixation allows more sensitive detection of signals. 6. Wash with PBS 5min, 3 times. 7. Trim blocks with a razor blade, blot dry blocks on Kimwipe paper, glue blocks to the specimen disc with superglue, tissue facing up. Keep blocks to cut on ice. 8. Filled the buffer tray with ice-cold PBS to the rim of the block, but not cover the block. Fill the cooling chamber with ice. Keeping all solutions and blocks cold help cutting sections of consistent thickness. 9. Cut with low speed (4.5) and high vibration frequency (10, maximum) at 75um. Note: Different thickness of 50um, 75, and 150um were tested to find the thickest thickness that allows sensitive detection of expression. 150um thickness reduces probe penetration, while 75um allow sensitive detection of both cortical and medullary signals when tested with BF2 and Wnt7b probes. 10. Collect the sections in PBS in 24-well plates. 11. Dehydrate the sections as with tissues for wholemount in situ hybridization and store in 100% Methanol. Note: microwave gelatin to dissolve it in PBS, be careful not to boil over. Store the stock at 4C. When making many blocks, keep the melted gelatin warm in 55C water bath. | McMahon Group | ||||
| RNA Isolation Procedure from Flow Sorted Cells | RNA extraction | TRIzol-LS, Invitrogen (Cat# 10296-010), Ambion Glycogen, Invitrogen (Cat# AM 9510), RNeasy Micro Kit, Qiagen (Cat# 74004), Isopropanol, DNAse I, Ambion (Cat# AM 2222), SUPERase•In, Ambion (Cat# AM 2694), Nuclease Free H2O, Ambion (Cat# AM 9930), B-Mercaptoethanol (BME), Sigma (Cat# M-7522), poly-d-inosine (pI), Epicentre Cat# PI6052H | 1. FACs sort cells directly into 0.75 ml of TRIsol-LS. After flow sort adjust to 1 ml. Do not exceed 1 ml volume 2. Add 50 µl Ambion Glycogen to the TRIsol-LS/cell lysate. Vortex thoroughly! 3. Add 200 µl CHCl3 and vortex thoroughly then shake for 15 seconds. 4. Let mixture sit for 5 minutes at room temperature so phases begin to separate. 5. Centrifuge at 12.2k RCF at 4°C for 10 minutes. 6. Remove 550 µl of upper clear aqueous and add directly into 550 µl of cold isopropanol. 7. Invert tube to mix and let sit on ice for 15 minutes. 8. Centrifuge to pellet RNA for 12.2k RCF at 4°C for 15 minutes. 9. After centrifugation small pellet should be visible at bottom of tube. Aspirate off the aqueous with a 1ml pipette tip. 10. Wash pellet with 1ml of ice cold 80% ethanol (made with DEPC H2O): centrifuge for 15 minutes 4°C, 7.5k RCF. 11. Air dry pellet for 2-3 minutes. 12. Resuspend pellet in 30 µl Ambion Nuclease free H2O and keep on ice. 13. Add reagents for DNase I digestion, pipette gently up/down to mix after final addition of enzyme. 30 µl RNA pellet dissolved in Ambion Nuc-free H2O, 3.7 µl DNase 10X Buffer, 2 µl RNasin inhibitor (SUPERase•In), 2 µl Ambion DNase1, 37.7µl is final volume 14. Incubate at 37°C for 25-30 minutes in a heat block, then store at -80°C until can proceed with subsequent column purification or proceed directly to column purification. 15. Thaw DNase-treated samples on ice. While thawing make up RLT solution (RLT/BME/pI is made up using 98.5 µl RLT, 1µl BME, and 0.5 µl pI ). 16. Add 100 µl of RLT/BME/pI to the DNAsed sample and pipette well up/down to mix. 17. Add 100 µl 70% ethanol (made with DEPC water) and mix well. 18. Transfer to a Qiacolumn and place into a collection tube. Let bind for 5 minutes. 19. Spin 5 minutes at 5,000 rpm (at RT). 20. Collect flow-through and reapply to column. Let bind for 5 minutes. 21. Spin 1 minute at 8,000 rpm. 22. Add 700 µl RW1 to the column and place into a new collection tube. 23. Spin 30 sec at 8,000 rpm. 24. Add 500 µl RPE to the column and place into a new collection tube. 25. Spin 30 sec at 8,000 rpm. 26. Add 500 µl RPE to the column (for a second wash) and place in a new collection tube. 27. Spin 30 sec at 8,000 rpm. 28. Add 500 µl 80% ethanol (made with DEPC-treated water) to the column and place into a new collection tube. 29. Spin 2 minutes at 8,000 rpm. Make sure the cap to the column is closed. 30. Place column into a new collection tube. 31. Spin 5 minutes 8,000 rpm with the cap open; if the cap closes during the spin, give it another 2 minute spin at 8,000 rpm. 32. Place column in 1.5 ml RNase-free eppendorf tube and add 30 µl RNase-free water (from Qiagen kit) to the column. Let sit for 2 minutes. 33. Spin 5 minutes at 5,000 rpm and then 30 seconds at 10,000 rpm. 34. Add another 30 µl RNase-free water and let sit for 2 minutes. 35. Spin for 5 minutes at 5,000 rpm and then 30 sec at 10,000 rpm. 36. Measure final volume: it should be equal to input volume to column 37. Aliquot 2 µl for QC/QA on Nanodrop and Bioanalyzer ‘pico’ chip to check integrity. | Southard-Smith Group | Harding SD Armit C Armstrong J Brennan J Cheng Y Haggarty B Houghton D Lloyd-MacGilp S Pi X Roochun Y Sharghi M Tindal C McMahon AP Gottesman B Little MH Georgas K Aronow BJ Potter SS Brunskill EW Southard-Smith EM Mendelsohn C Baldock RA Davies JA Davidson D. The GUDMAP database – an online resource for genitourinary research. Development. 2011 Jul; 138(13):2845-53. McMahon AP Aronow BJ Davidson DR Davies JA Gaido KW Grimmond S Lessard JL Little MH Potter SS Wilder EL Zhang P. 2008. GUDMAP: the genitourinary developmental molecular anatomy project. J Am Soc Nephrol. 19 667-671. | ||
| In situ hybridisation on vibrating microtome-cut mouse tissue sections | Make sample baskets | 1. Use heated razor blade to cut off bottom of microcentrifuge tube (at or just below 100mL mark). 2. Cut polyester mesh into squares that are large enough to cover the cut part of the microfuge tube. 3. Heat cut edge of tube in flame until softened. 4. Press melted surface of microfuge tube firmly onto center of mesh square & hold for a few seconds to seal the mesh along entire circumference of tube. 5. Trim away excess mesh with scissors (may also heat near flame to melt down excess). | Vezina Group | ||||
| Sectioning | Tissue sectioning | cryostat, microdissection instrument | poly-lysine solution, | Use the cryostat in Prasad’s lab. Place mold in the chamber for a few minutes to temperature equilibrate. Remove tinfoil. Place chuck that has been at room temp in chamber and let cool a minute, but not too much. Place OCT on chuck and let cool a minute, but not freeze, and then place tissue OCT block on chuck, and let freeze in position. Can place additional OCT around the base and spread with gloved finger to help hold in place. The tissue is too brittle to section properly if the chamber is too cold. I typically use a setting for the chamber and arm of –12 to –14. We have been using –14 mostly lately, with success. Use the trim setting of 40-60 to remove most of excess OCT, til see tissue. When getting close to tissue can drop back from trim to regular setting, of 9 microns for Veritas, with UV cutting, or 7 microns for old Pixcell II machine. Use a regular glass slide to check for presence of tissue in sections. When you hit a good chunk of tissue start saving on the membrane slides (see below). Membrane slides are prepared as follows to allow good sticking of the sections to the slides. Dilute the Sigma poly-lysine solution one to ten, as recommended by Sigma. Dip slides and dry in vertical position. Need to carve the OCT block with a razor blade, so the tissue sections are not too large. Be very, very careful here not to cut yourself on the blade in the cryostat, which is very sharp. Collect sections on the slides. Do not want the tissue sections to be arranged so that they end up under the strut supports of the caps, as this would give cap crap. So the sections should be spaced so that the cap can rest with one tissue section centered, and with no other tissue section under the edges. So space them out, and try to get 5-10 sections per membrane slide. Try to work fairly fast, as the RNA can go bad sitting in one section at room temp while other sections are being cut on the slide. Try to get the best sections possible. Sometimes having the cut part go more slowly helps. Sometimes warming a degree or two helps. Sometimes changing the way you catch the section with the brushes as it comes off of the blade helps. Place the slides with sections in box with dry ice against the slide, and then store at –80ºC. | Potter Group | ||
| Optimized Vibratome Section in situ Hybridization for P1 Mouse Kidney Sections | wash | 1. Wash 3x with MBST for 5' at RT (make up 1000 mls + 2 mM Levimasole). 2. Wash 10x with MBST for ??1 hr each at RT. 3. Wash 8x with MBST for ???2hr each at 4C. 4. Wash 3x with NTMT for 5' at RT (make up 60 ml + 2 mM Levimasole). 2.0 ml 5 M NaCl 5.0 ml 2 M Tris 9.5 5.0 ml of 1 M MgCl2 0.1 ml tween -20 100 ml 5. Transfer sections to 48-well tissue culture plates and then replace NTMT with 0.5ml BM Purple (Roche 1 442 074 ). Wrap in foil, monitor signal every few hrs, usually takes from 6hrs to 48hrs. Note: Shh, a weakest probe, produces signals after 48hrs of color reactions with acceptable level of background. So 48hrs was chosen as the longest time of color development. 6. When reaction is complete; wash 3x in PBT, *pH 4.5,(pH PBS with HCl), for 5 RT, keep in dark. 7. Fix in 4% paraformaldehye plus 0.1% glutaraldehyde, for 1 hr-o/n. 8. Wash in 50% then 80% glcerol/PBS. Mount slides on VWR precleaned superfrost glass slides. Remove excess glycerol with Wattman paper. Mount coverslip using Glycergel. (*Need to drop pH of PBT to 4.5 to stop reaction completely, also whitens up sections) | McMahon Group |
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