The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation

The effect of three proteases--trypsin, pepsin, and pronase--on the immunohistochemical staining of keratins with a broad-spectrum monoclonal antibody was investigated in paraffin sections of formalin and ethanol-fixed tissues by means of the peroxidase-antiperoxidase method. Both the length of exposure to the fixative and the duration of proteolysis were varied over a wide range. Ethanol-fixed tissues showed excellent preservation of the antigenicity of keratins, and no appreciable differences in immunostaining related to the length of fixation were found. The use of proteolytic enzymes did not improve these results; on the contrary, it caused rapid tissue disintegration. Formalin-fixed epithelial tissues stained weakly or failed to stain unless they were treated with a proteolytic enzyme. The optimal length of proteolysis varied with the degree of fixation; tissues that were fixed for long periods of time in formalin required longer exposure to a proteolytic enzyme and were more resistant to digestion than were tissues that were fixed briefly. No significant advantage of one protease over another was found in this study. We conclude that a proteolytic step must precede immunostaining for keratins if the tissue is fixed in formalin, but that the digestion period must be adjusted according to the length of exposure to the fixative. The superiority of alcohol over formalin fixation for the preservation of the antigenicity of keratins is confirmed by this study.     

Polyester wax embedding and sectioning technique for immunohistochemistry

We have developed a method useful for immunohistochemical studies by combining tissue fixation with buffered neutral formalin and polyester wax embedding. Buffered neutral formalin fixation preserves cell and tissue fine structure, and also the antigenicity of unstable enzymes. Polyester wax embedding makes possible thin serial sections of various tissues and preserves antigenicities for at least 6 months. We have demonstrated using this technique the localization of alpha-amylase in mouse salivary gland, parietal-cell specific antigen in mouse glandular stomach, and DNA polymerase alpha and beta in chick tissue.     

Application of microwave irradiation to immunohistochemistry: preservation of antigens of the extracellular matrix

Microwave irradiation as a means of fixation was evaluated for the preservation of extracellular matrix antigens such as collagen III, IV, fibronectin and laminin in both lung and liver specimens. Small tissue samples were placed in normal saline or periodate-lysine-paraformaldehyde (PLP) and irradiated for 30 sec to bring them to a temperature of 50 C. The tissue was then processed rapidly in a tissue processor adjusted to a 2 hr cycle and embedded in paraffin. Sections were immunostained. For comparison, routine cryostat sections as well as sections of formalin fixed tissue were used. Microwave irradiation in saline gave excellent morphological detail, comparable to that in formalin fixed tissue. All four antigens evaluated were well preserved without the necessity of prior pepsin digestion. Microwave fixation is promising for preservation of antigenicity and morphological detail, and considerably reduces the time required for processing.     

Application of Microwave Irradiation to Immunohistochemistry: Preservation of Antigens of the Extracellular Matrix

Microwave irradiation as a means of fixation was evaluated for the preservation of extracellular matrix antigens such as collagen III, IV, fibronectin and laminin in both lung and liver specimens. Small tissue samples were placed in normal saline or periodatelysine-paraformaldehyde (PLP) and irradiated for 30 sec to bring them to a temperature of 50 C. The tissue was then processed rapidly in a tissue processor adjusted to a 2 hr cycle and embedded in paraffin. Sections were immunostained. For comparison, routine cryostat sections as well as sections of formalin fixed tissue were used. Microwave irradiation in saline gave excellent morphological detail, comparable to that in formalin fixed tissue. All four antigens evaluated were well preserved without the necessity of prior pepsin digestion. Microwave fixation is promising for preservation of antigenicity and morphological detail, and considerably reduces the time required for processing.     

One-step preservation of phosphoproteins and tissue morphology at room temperature for diagnostic and research specimens

Background

There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block.

Results

Total protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79.

Conclusion

In a single paraffin block BHP preserved the phosphorylation state of several signaling proteins at a level comparable to snap-freezing, while maintaining the full diagnostic immunohistochemical and histomorphologic detail of formalin fixation. This new tissue fixative has the potential to greatly facilitate personalized medicine, biobanking, and phospho-proteomic research.     

Influence of smear preparation and fixatives on the DNA ploidy and the morphonuclear features of the MXT mammary tumor and normal tissues in the mouse

We compared cytomorphonuclear parameters--related to DNA histogram and chromatin distribution--on MXT mouse mammary tumor and murine normal cells from fresh squash smears or from deparaffinized tissue smears fixed in several fluids. We used the SAMBA 200 cell image processor with software allowing for the discrimination of parameters computed on Feulgen-stained nuclei. The spectrophotometric results--assessed by integrated optical density values--indicate that the nuclei from deparaffinized tissue fixed in Bouin's fluid are around 50% less stained than those fixed in formalin or ethanol-formalin-acetic acid (EFA). The fresh smears of nuclei fixed in formalin contain a less well-defined and more homogeneous chromatin than after Bouin's or EFA fixation. This has led to the conclusion that morphonuclear parameter comparisons performed on tissues differently processed or from different origins present severe limitations.     

The importance of tissue fixation for light microscopic immunohistochemical localization of peroxisomal proteins: the superiority of Carnoy's fixative over Baker's formalin and Bouin's solution

We have compared the effects of fixation with three commonly used fixatives upon preservation of the antigenicity of six peroxisomal proteins in rat liver using both immunohistochemical staining and Western blotting of fixed tissue extracts. The immunoreactivity of all six peroxisomal proteins was well preserved and peroxisomes were clearly identified in material fixed in Carnoy's fixative. Moreover, the corresponding proteins stained well in Western blots prepared from extracts of Carnoyfixed material. The intensity of the immunohistochemical staining was reduced at different rates for individual peroxisomal proteins after fixation in Baker's formalin, but peroxisomes were still well visualized with antibodies to catalase and some -oxidation enzymes. No evidence of immunohistochemical staining for any peroxisomal antigens was obtained after fixation in Bouin's fluid. For detection of the antibody binding sites in Carnoy's fixed material, the avidin-biotin-peroxidase complex (ABC) with aminoethyl carbazole as chromogen was found to be superior to the methods of peroxidase-antiperoxidase/diaminobenzidine and protein A-gold with silver intensification. Using Carnoy-fixative and the ABC-method, we demonstrate light microscopic immunohistochemical localization of peroxisomal antigens in several rat tissues as well as in human post-mortem liver.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

HistoChoice as an alternative to formalin fixation of undecalcified bone specimens.

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visualized more easily in HistoChoice fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Simultaneous detection of EGFP and cell surface markers by fluorescence microscopy in lymphoid tissues.

Enhanced GFP (EGFP) is a powerful tool for the visualization of tagged proteins and transfected cells and is easily detected by fluorescence microscopy or flow cytometry in living cells. However, soluble EGFP molecules can be lost if cell integrity is disrupted by freezing, sectioning, or permeablization. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilize EGFP may also destroy its usefulness as a fluorescent reporter. Here we determined which methods of preparing murine lymphoid tissues immobilized soluble EGFP protein and retained its fluorescence while simultaneously maintaining the antigenicity of various immunologically important molecules and best preserving the overall morphology of the tissues. We found that EGFP could not be visualized in frozen sections of spleen that had not been fixed before freezing. However, robust EGFP fluorescence could be observed in frozen sections of tissues fixed under various conditions. Fixation was important to immobilize EGFP rather than to maintain conformation, because only minimal EGFP could be detected by immunofluorescence in unfixed frozen sections. Although it had little effect on EGFP fluorescence, the inclusion of sucrose during fixation better preserved the morphology of fixed tissues. These methods also preserved the antigenicity of a wide variety of molecules used to identify cell types in lymphoid tissues.     

Impact of tissue preservation on collagen fiber architecture

Microarchitectural features of collagen-rich extracellular matrices provide the mechanical foundation for tissue function and exhibit topographical cues that influence cellular behavior including proliferation, migration and protein expression. Preservation of tissue microarchitecture is required for accurate evaluation of tissue characteristics and pathology. It is unclear whether common tissue preservation methods possess equal ability to preserve microarchitecture. We investigated collagen microarchitecture in samples that had been flash frozen, fixed in formalin or preserved in RNAlater®, and which contained both collagen-rich and collagen-sparse regions. Fibrillar collagen organization was characterized using picrosirius red staining and second harmonic generation (SHG) microscopy. Maintenance of collagen fiber characteristics compared to the gold standard of flash freezing depended on both the method of preservation and the local collagen content of the tissue. Both formalin fixation and RNAlater® preserved collagen fiber characteristics similar to flash freezing in collagen-rich areas of the tissue, but not in collagen-sparse regions. Analysis using picrosirius red staining indicated preservation-dependent changes in overall tissue architecture and suprafibrillar organization. Together with considerations of cost, ease of use, storage conditions and ability to use the preserved tissue for RNA or protein analysis, our quantitative characterization of the effects of preservation method on collagen microarchitecture may help investigators select the most appropriate preservation approach for their needs.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Preferential nuclear binding of estrogen in the formalin-fixed rat uterus

Rat uterus fixed overnight in buffered formalin retains the ability to specifically bind estradiol. However, the estrogen binding property of fixed tissue appears preferentially localized in the nuclear fraction regardless of hormonal status. Furthermore, the quantity of the nuclear estrogen receptor in fresh or fixed uterus is virtually identical in the presence or absence of estrogenic hormone. Yet, while both tissue preparations exhibit equivalent increases in the total nuclear receptor occupancy after hormone exposure, only the fresh uterus contains a major cytosolic estrogen binder which decreases in availability upon the estrogen-induced elevation of the nuclear bound steroid. However, the cytosolic estrogen receptor exhibits a significant loss in its ligand binding property after formalin exposure. Thus, the preferential localization of estrogen binding in the nuclear fraction of fixed whole tissue may just reflect that only the tightly bound nuclear estrogen receptor's functional and/or structural integrity survives long-term formation fixation. Our observation of estrogen binding in preserved tissue may also be a clinically useful tool in therapy analysis.     

Histological assessment of PAXgene tissue fixation and stabilization reagents

Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities.     

固定对组织光学性质的影响

利用带有积分球的SHIMADZU UV—240分光光度计,测量了组织在固定前和后的反射率和透射率,分析了福尔马林固定对组织光学性质的影响。     

Chemical and Spectrometric Evaluation of Glycogen After Routine Histological Fixatives

A detailed comparison of fixatives used for the demonstration of glycogen has been based on chemical assay and microspectrophotometry. Rat liver containing known amounts of glycogen was fixed in formol alcohol, Rossman's fluid, 10% neutral formalin, Bouin, Helly, SUSA, and Zenker's fluid at 4 C and 18 C. Chemical assay was carried out before and after fixation and paraffin sections were prepared from the fixed material. Sections were stained with PAS and the silver methenamine method. Visual examination was carried out with a comparison microscope and quantitative estimations on PAS-stained sections were performed by scanning microspectrophotometry. The histochemical methods were compared with the chemical results obtained from the same tissue and a reasonable degree of correlation between the sets of results was observed. Cold formol alcohol and cold Rossman's fluid preserved the most glycogen and Zenker and SUSA fixation preserved the least. Cold formol alcohol was the only fixative that preserved threshold values of glycogen i.e. 0.3% and the silver methenamine method is recommended for the demonstration of these small amounts.     

Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues

Fresh or frozen tissue samples will always be the best tissue source for the analysis of nucleic acids and proteins from tissues. However, their long-term storage is expensive and laborious. Much interest has therefore been focused on the question whether the almost infinite resources of formalin fixed and paraffin embedded tissue samples in the archives of pathology and histology departments can be used for research on biomarkers and molecular mechanisms of disease. In recent years the methods and protocols for the extraction of DNA, mRNA, miRNA and proteins from formalin-fixed and paraffin-embedded tissue samples have improved enormously. Especially, the possibilities of analysing DNA and miRNA in FFPE have reached a level that allows their application as a first line approach in the search for biomarkers. In contrast, many questions remain in terms of quantification of mRNA and protein expression levels in formalin-fixed and paraffin-embedded tissue samples. This review gives an overview on current potentials and limitations of the quantification of DNA, miRNA, mRNA and the proteome in FFPE tissue samples. The chemical events during formalin fixation and paraffin embedding and alternatives to formalin fixation are described. In addition, methods and general problems of DNA, miRNA, mRNA and protein extraction and the current knowledge on the feasibility and accuracy of quantitative gene expression analysis in FFPE tissues is summarized.     

Immunohistochemical localization of zona pellucida proteins ZPA, ZPB and ZPC in human, cynomolgus monkey and mouse ovaries

The zona pellucida of mammalian oocytes plays an important role in binding and activation of sperm cells during the molecular events leading to fertilization. The genes coding for the three zona pellucida glycoproteins ZPA, ZPB, and ZPC of various species including mouse, dog, and human have been cloned and sequenced by several groups. However, it has remained a matter of debate as to whether the oocytes alone or in conjunction with the surrounding granulosa cells express and deposit these proteins to form the zona pellucida matrix. Addressing this unresolved issue, we assessed the expression and localization of all three zona pellucida proteins in ovaries of human, cynomolgus monkey and mice using immunohistochemical methods. In addition, oocyte-specific expression of ZPC from the primordial stage onward was confirmed by in situ hybridization. In sections of human ovaries, ZPA, ZPB, and ZPC proteins were immunohistochemically detected in the cytoplasms of primordial oocytes and during later stages of folliculogenesis in the zona pellucida matrices of oocytes. In sections fixed with formalin, a clear homogeneous ring was visible around the oocyte and no staining of granulosa cells was observed. In contrast, staining of ZP proteins was also observed between granulosa cells when Bouin's reagent had been used for tissue fixation. Thus, the original zona pellucida architecture was better preserved by formalin fixation. We further demonstrated that dissolution of the zona pellucida of isolated bovine oocytes occurred after they were exposed to Bouin's reagent. In summary, these results demonstrate that in mice, monkeys and humans, zona proteins are expressed and assembled exclusively by the oocyte and not by the granulosa cells. Previously observed results of ZP expression by an involvement of granulosa cells might therefore be the result of an improper fixation of the tissues leading to the disruption of the zona pellucida. Additionally this study highlights the importance of choosing the correct fixative for immunohistochemistry, not only for the usual reason of retaining antigenicity, but rather to retain the entire architectural structure.     

Special symposium: fixation and tissue processing models

Abstract

Fixation and processing of tissue to paraffin blocks permit thin (4-5 µm) sections of tissues to be cut. Tissues and their subcellular components and surrounding stroma are visualized by cutting thin sections and staining them histochemically or immunohistochemically and viewing the sections using a bright field microscope. During the last century, anatomists and pathologists have used fixation with 10% neutral buffered formalin (10% NBF) as the fixative of choice. Also, both human and veterinary pathologists have trained to use fixation with 10% NBF, so these professionals are reluctant to change the familiar microscopic appearance of diagnostic tissues by using different fixatives. In addition, the effects of tissue processing on the microscopic appearance of tissue essentially has been ignored in most studies. Archives of paraffin blocks of pathological tissue contain essentially paraffin blocks fixed in 10% NBF. Therefore, if retrospective studies use archival paraffin blocks to correlate the molecular features of diseases with their outcomes, the studies must be based on tissue fixed in 10% NBF. Studies of how fixation in 10% NBF interacts with histochemical and immunohistochemical staining are limited in number and most are based on relatively long fixation times (≥36 h). Currently, fixation times in 10% NBF have been reduced to <24 h. Little is known about fixation in 10% NBF and its interaction with tissue processing for any period of fixation, especially short times. Less is known about how fixation of tissues with 10% NBF interacts with more modern assays using immunohistochemistry, real time quantitative polymerise chain reaction (PCR), and techniques that depend on analysis of proteins extracted from paraffin blocks including multiplex immunoassays or mass spectrometry. In general, multiple antibody–antigen combinations are reported not to work in tissues fixed in 10% NBF, i.e., loss of immunorecognition is nearly complete for such antibody–antigen combinations as Ki67/MIB, estrogen receptor alpha (ERα) and Progesterone receptor (PR), and partial for Bcl-2. Several models have been developed to study the interactions of tissue fixation and immunorecognition, but most have viewed the problem with immunorecognition as completely caused by fixation. Also, some of the models discussed in this special symposium do not predict the effects of fixation on frozen tissues fixed in 10% NBF and not processed to paraffin blocks. This article is a brief review of issues attending the use of 10% NBF combined with tissue processing as an interrelated process to study biomarkers identified by immunohistochemistry.