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Paraffin embedding is widely used in microscopic imaging for preparing biological specimens. However, owing to significant fluorescence quenching during the embedding process, it is not compatible with fluorescent-labeling techniques, such as transgenic and viral labeling using green fluorescent protein (GFP). Here, we investigate the quenching mechanism and optimize the embedding process to improve the preservation of fluorescence intensity. The results show that dehydration is the main reason for fluorescence quenching during paraffin embedding, caused by the full denaturation of GFP molecules in ethyl alcohol. To evaluate fluorescent and morphological preservation, we modified the embedding process using tertiary butanol (TBA) instead of ethyl alcohol. Fluorescence intensity following TBA dehydration increased 12.08-fold of that observed in the traditional method. We obtained uniform fluorescence maintenance throughout the whole mouse brain, while the continuous apical dendrites, spines, and axon terminals were shown evenly within the cortex, hippocampus, and the amygdala. Moreover, we embedded a whole rat brain labeled with AAV in the prelimbic cortex (Prl). With the axon terminals in different areas, such as the caudate putamen, thalamus, and pyramidal tract, the results showed a continuous tract of Prl neurons throughout the whole brain. This method was also suitable for tdTomota labeled samples. These findings indicate that this modified embedding method could be compatible with GFP and provides a potential turning point for applications in the fluorescent labeling of samples.
Paraffin histology is one of the most important and commonly used laboratory techniques in diagnostic histopathology. The discovery of paraffin embedding is often attributed to the pathologist Edwin Klebs. Klebs was following the lead of Stricker, who embedded embryos in a mixture of hot stearin and white beeswax. We show that Klebs experimented with paraffin wax for embedding tumour tissue. But he quickly rejected it as unsuitable because paraffin wax did not infiltrate the tissue. One of Klebs' correspondents, embryologist Wilhelm His, Sr., learned of Klebs' experiments and decided to try paraffin embedding. His dehydrated chicken embryos in alcohol, cleared them in lavender oil, and dripped hot paraffin wax onto them. This process allowed His to cut good sections. Here, we have replicated His's paraffin embedding protocol in order to determine whether His had indeed made the landmark discovery of infiltration embedding with paraffin wax. We followed the protocol that he gives in his 1868 monograph on the early development of the chicken. The protocol described by His failed, in our hands, to yield sections of the quality that he illustrates in his monograph. Typically, the tissue disintegrated when sectioned due to poor infiltration of the wax. Usable sections could only be obtained if His's protocol was modified by melting the embedded embryos in fresh paraffin wax. One explanation for our findings is that we failed to faithfully replicate His's protocol. Another is that his protocol was incomplete. We suggest that His is likely to have discovered and perfected infiltration embedding with paraffin wax but did not publish a complete protocol.
We have developed a straightforward method that uses paraffin-embedded bone for undemineralized thin sectioning, which is amenable to subsequent dynamic bone formation measurements. Bone has stiffer material properties than paraffin, and therefore has hereforto usually been embedded in plastic blocks, cured and sectioned with a tungsten carbide knife to obtain mineralized bone sections for dynamic bone formation measures. This process is expensive and requires special equipment, experienced personnel, and time for the plastic to penetrate the bone and cure. Our method utilizes a novel way to prepare mineralized bone that increases its compliance so that it can be embedded and easily section in paraffin blocks. The approach is simple, quick, and costs less than 10% of the price for plastic embedded bone sections. While not effective for static bone measures, this method allows dynamic bone analyses to be readily performed in laboratories worldwide which might not otherwise have access to traditional (plastic) equipment and expertise.
Combining histology and ex vivo MRI from the same mouse brain is a powerful way to study brain microstructure. Mouse brains prepared for ex vivo MRI are often kept in storage solution for months, potentially becoming brittle and showing reduced antigenicity. Here, we describe a protocol for mouse brain dissection, tissue processing, paraffin embedding, sectioning, and staining. We then detail registration of histology to ex vivo MRI data from the same sample and extraction of quantitative histological measurements.
The enzyme-labeled antigen method is an immunohistochemical technique detecting plasma cells producing specific antibodies in tissue sections. The probe is an antigen labeled with an enzyme or biotin. This immunohistochemical technique is appliable to frozen sections of paraformaldehyde (PFA)-fixed tissues, but it has been difficult to apply it to formalin-fixed, paraffin-embedded (FFPE) sections. In the current study, factors inactivating the antibody reactivity during the process of preparing FFPE sections were investigated. Lymph nodes of rats immunized with horseradish peroxidase (HRP) or a mixture of keyhole limpet hemocyanin/ovalbumin/bovine serum albumin were employed as experimental models. Plasma cells producing specific antibodies, visualized with HRP (as an antigen with enzymatic activity) or biotinylated proteins in 4% PFA-fixed frozen sections, significantly decreased in unbuffered 10% formalin-fixed frozen sections. The positive cells were further decreased by paraffin embedding following formalin fixation. In paraffin-embedded sections fixed in precipitating fixatives such as ethanol and acetone and those prepared with the AMeX method, the antigen-binding reactivity of antibodies was preserved. Fixation in periodate-lysine-paraformaldehyde and Zamboni solution also kept the antigen-binding reactivity in paraffin to some extent. In conclusion, formalin fixation and paraffin embedding were major causes inactivating antibodies. Precipitating fixatives could retain the antigen-binding reactivity of antibodies in paraffin-embedded sections.
Elucidation of molecular pathways involved in development of human lymphoma requires efficient methods for tackling gene expression in lymph nodes. Expression studies of transcription factors in these malignancies facilitate understanding the changes occurring in neoplastic transformation and lymphoma development. Excised lymph nodes are routinely fixed in formalin and embedded in paraffin for diagnosis and stored in many hospitals' pathology archives. These tissues represent a precious resource for research since they allow retrospective studies to cover a broad range of human lymphoma even the less frequent types. Reverse transcription polymerase chain reaction (RT-PCR) is a commonly used method for gene expression analysis and a reproducible protocol for RNA isolation from lymph nodes is an inevitable requirement for these studies. However, formalin fixation and paraffin-embedding interfere with the quality of RNA especially when isolated from lymph nodes being the most fragile lymphatic tissues. We present here a simple and fast method for RNA isolation from formalin-fixed paraffin-embedded lymph nodes that can be successfully applied for RT-PCR as well as for quantitative RT-PCR analysis. We tested diverse isolation reagents and combined a range of factors in order to get a high quality RNA for retrospective studies of gene expression in human lymphoma samples. Our modified method of RNA extraction from FFPE provides superior yields and purity based on qPCR data.
Intranuclear birefringent inclusions (IBI) found in various cell types in paraffin-embedded tissue sections have long been considered to be a tissue processing artifact, although an association with biological processes has been suggested. We applied polychromatic polarization microscopy to image their spatial organization. Our study provides evidence that IBI are caused by liquid paraffin-macromolecular crystals formed during paraffin-embedding procedures within cells and potentially reflect an active transcriptional status.
In clinical practice, patients' tissues are fixed and paraffin-embedded in order to enable histological diagnosis. Nowadays, those tissues are also used for molecular characterization. Formalin is the most used fixative worldwide, and Bouin's solution in some worldwide institutions. Among molecular targets, micro RNAs (miRNAs), the single-stranded non-coding RNAs comprised of 18 to 24 nucleotides, have been demonstrated to be resistant to fixation and paraffin-embedding processes, with consequent possible application in clinical practice. In the present study, let-7e-5p, miR-423-3p, miR-92a-1-5p, miR-30d-5p, miR-155-5p, miR-200a-3p, and miR-429 were investigated in formalin and matched Bouin's solution-fixed tissues of high grade serous ovarian cancers by means of real-time and droplet digital PCR (ddPCR). Micro RNAs were detectable and analyzable in both formalin- and Bouin's-fixed specimens, but on average, higher Ct values and lower copies/µL were found in Bouin's-fixed samples. Data from formalin-fixed samples correlated significantly for most targets with Bouin's ones, except for let-7e-5p and miR-155-5p. This study shows that miRNAs are analyzable in both formalin- and Bouin's-fixed specimens, with the possibility, after proper data normalization, to compare miRNA-based data from formalin-fixed samples to those of Bouin's-fixed ones.
Standard histopathology techniques (including paraffin embedding) are incompatible with thick tissue multiphoton imaging, and standard clearing techniques on those specimens destroy some molecular information. We demonstrate multiphoton imaging in specimens prepared according to standard histopathology techniques. This permits unlabeled 3-dimensional histology on archival tissue banks, which is of great value in evaluating prognostic indicators.
Three-dimensional (3D) cell culture models such as spheroids and organoids are widely used in the field of experimental biology. To analyze these 3D experimental models, formalin-fixed paraffin-embedded (FFPE) sections are superior to whole-mount imaging for some experimental purposes, such as exploring samples with a depth limitation of primary antibody penetration immunohistochemically. However, tiny 3D cell culture samples are difficult to embed in paraffin and acquire appropriate sections. In this report, we optimized a protocol of paraffin embedding for spheroids and organoids. In addition, we compared FFPE sections with frozen sections in ratio of sample collection and section condition after staining, and could reproduce improved results reliably. The protocol we established could be widely used in many laboratories and become a useful technique for analyzing spheroids and organoids.
In recognition of the importance of zebrafish as a model organism for studying human disease, we have created zebrafish content for a web-based reference atlas of microanatomy for comparing histology and histopathology between model systems and with humans (http://bio-atlas.psu.edu). Fixation, decalcification, embedding, and sectioning of zebrafish were optimized to maximize section quality. A comparison of protocols involving six fixatives showed that 10% Neutral Buffered Formalin at 21°C for 24h yielded excellent results. Sectioning of juveniles and adults requires bone decalcification; EDTA at 0.35M produced effective decalcification in 21-day-old juveniles through adults (≥~3Months). To improve section plane consistency in sets of larvae, we have developed new array casting molds based on the outside contours of larvae derived from 3D microCT images. Tissue discontinuity in sections, a common barrier to creating quality sections of zebrafish, was minimized by processing and embedding the formalin-fixed zebrafish tissues in plasticized forms of paraffin wax, and by periodic hydration of the block surface in ice water between sets of sections. Optimal H&E (Hematoxylin and Eosin) staining was achieved through refinement of standard protocols. High quality slide scans produced from glass histology slides were digitally processed to maximize image quality, and experimental replicates posted as full slides as part of this publication. Modifications to tissue processing are still needed to eliminate the need for block surface hydration. The further addition of slide collections from other model systems and 3D tools for visualizing tissue architecture would greatly increase the utility of the digital atlas.
Formalin-fixed paraffin-embedded (FFPE) tissue specimens constitute a highly valuable source of clinical material for retrospective molecular studies. However, metabolomic assessment of such archival material remains still in its infancy. Hence, there is an urgent need for efficient methods enabling extraction and profiling of metabolites present in FFPE tissue specimens. Here we demonstrate the methodology for isolation of primary metabolites from archival tissues; either fresh-frozen, formalin-fixed or formalin-fixed and paraffin-embedded specimens of mouse kidney were analysed and compared in this work. We used gas chromatography followed by mass spectrometry (GC/MS approach) to identify about 80 metabolites (including amino acids, saccharides, carboxylic acids, fatty acids) present in such archive material. Importantly, about 75% of identified compounds were detected in all three types of specimens. Moreover, we observed that fixation with formalin itself (and their duration) did not affect markedly the presence of particular metabolites in tissue-extracted material, yet fixation for 24h could be recommended as a practical standard. Paraffin embedding influenced efficiency of extraction, which resulted in reduced quantities of several compounds. Nevertheless, we proved applicability of FFPE specimens for non-targeted GS/MS-based profiling of tissue metabolome, which is of great importance for feasibility of metabolomics studies using retrospective clinical material.
Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.
Large repositories of well characterized RNAlater preserved samples and formalin-fixed, paraffin-embedded samples have been generated worldwide. However, the impact on the proteome of the preservation methods remain poorly described. Therefore, we analyzed the impact on the proteome of preserving samples in RNAlater, and by formalin-fixation, paraffin-embedding on human soft tissue, using directly frozen samples as a control ("Comparing the proteome of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human tissue samples" [1]). We here report the data from the analysis. The comparative analysis was performed on 24 colon mucosa biopsies, extracted from the sigmoideum of two gastroenterologically healthy participants for the purpose of this study. A set of biopsies were additionally stored for 30 min at room temperature prior to formalin-fixation. The samples were analyzed by high throughput gel free quantitative proteomics. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002029.
Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.
Formalin fixing with paraffin embedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing.
Neuron counting in the cochlea is a crucial but time-consuming operation for which various methods have been developed. To improve simplicity and efficiency, we tested an imaging method of the cochlea, and based on Confocal Laser Scanning Microscopy (CLSM), we visualised Rosenthal's Canal and quantified the spiral ganglion neurons (SGN) within. Cochleae of 8 normal hearing guinea pigs and one implanted with a silicone filament were fixed in paraformaldehyde (PFA), decalcified, dehydrated and cleared in Spalteholz solution. Using the tissue's autofluorescence, CLSM was performed at 100 fold magnification generating z-series stacks of about 20 slices of the modiolus. In 5 midmodiolar slices per cochlea the perimeters of the Rosenthal's Canal were surveyed, representative neuron diameters were measured and the neurons first counted manually and then software-assisted. For comparison, 8 normal hearing guinea pig cochleae were embedded in paraffin and examined similarly. The CLSM method has the advantage that the cochleae remain intact as an organ and keep their geometrical structure. Z-stack creation is nearly fully-automatic and frequently repeatable with various objectives and step sizes and without visible bleaching. The tissue shows minimal or no shrinking artefacts and damage typical of embedding and sectioning. As a result, the cells in the cleared cochleae reach an average diameter of 21 μm and a density of about 18 cells/10,000 μm(2) with no significant difference between the manual and the automatical counts. Subsequently we compared the CLSM data with those generated using the established method of paraffin slides, where the SGN reached a mean density of 9.5 cells/10,000 μm(2) and a mean soma diameter of 13.6 μm. We were able to prove that the semi-automatic CLSM method is a simple and effective technique for auditory neuron count. It provides a high grade of tissue preservation and the automatic stack-generation as well as the counter software reduces the effort considerably. In addition this visualisation technique offers the potential to detect the position and orientation of cochlear implants (CI) within the cochlea and tissue growing in the scala tympani around the CI and at the position of the cochleostomy due to the fact that the implant does not have to be removed to perform histology as in case of the paraffin method.
Spatial transcriptome analysis of formalin-fixed paraffin-embedded (FFPE) tissues using RNA-sequencing (RNA-seq) provides interactive information on morphology and gene expression, which is useful for clinical applications. However, despite the advantages of long-term storage at room temperature, FFPE tissues may be severely damaged by methylene crosslinking and provide less gene information than fresh-frozen tissues. In this study, we proposed a sensitive FFPE micro-tissue RNA-seq method that combines the punching of tissue sections (diameter: 100 μm) and the direct construction of RNA-seq libraries. We evaluated a method using mouse liver tissues at two years after fixation and embedding and detected approximately 7000 genes in micro-punched tissue-spots (thickness: 10 μm), similar to that detected with purified total RNA (2.5 ng) equivalent to the several dozen cells in the spot. We applied this method to clinical FFPE specimens of lung cancer that had been fixed and embedded 6 years prior, and found that it was possible to determine characteristic gene expression in the microenvironment containing tumor and non-tumor cells of different morphologies. This result indicates that spatial gene expression analysis of the tumor microenvironment is feasible using FFPE tissue sections stored for extensive periods in medical facilities.
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