Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue

We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.     

Effects of Prolonged Formalin Fixation on Diagnostic Immunohistochemistry in Domestic Animals

Immunohistochemistry (IHC) is routinely used in diagnostic pathology to detect infectious agents, to immunophenotype neoplastic cells, and to prognosticate neoplastic diseases. Formalin fixation is considered a limiting factor for IHC because formalin can cross-link antigens and mask epitopes. Prolonged formalin fixation is presumed to result in decreased antigen detection; however, this effect has only been evaluated with a few antibodies. The goal of this study was to evaluate the effect of prolonged formalin fixation on the immunohistochemical detection of 61 different antigens. Approximately 5-mm-thick tissue slices were fixed in 10% neutral-buffered formalin. Tissue slices were removed from formalin, processed, and paraffin-embedded at 1-day, 3-day, and then at ∼1-week intervals. IHC was performed on all sections in tandem after all tissues were processed. Immunoreactions were evaluated by three pathologists according to a four-tier grading system. Immunoreactivity of cytokeratin 7, high-molecular-weight cytokeratin, and laminin was diminished by prolonged formalin fixation. However, immunohistochemical reactivity remained moderate to strong with up to 7 weeks of fixation for all other antibodies. These results suggest that prolonged formalin fixation has minimal effects on antigen detection for most commonly used antibodies. These results further validate the use of IHC in diagnostic pathology. (J Histochem Cytochem 57:753–761, 2009)     

Leaching of Trace Elements from Biological Tissue by Formalin Fixation

In studies of trace elements in biological tissue, it is imperative that sample handling does not substantially change element concentrations. In many cases, fresh tissue is not available for study, but formalin-fixed tissue is. Formalin fixation has the potential to leach elements from the tissue, but few studies have been published in this area. The concentrations of 19 elements were determined by high-resolution inductively coupled plasma mass spectrometry in formalin in which human and rat brain samples had been stored for different time durations ranging from weeks up to several years. Additional analysis was carried out in fixed brain samples. There was substantial leaching of elements from the tissue into the formalin, and the leaching varied considerably between different elements. For example, formalin concentrations of As, Cd, Mg, Rb, and Sb increased more than 100-fold upon long-term (years) storage, while for Ni and Cr, the leaching was negligible. The degree of leaching was strongly time-dependent. In conclusion, formalin fixation and storage of biological tissue has the potential to leach substantial fractions of several trace elements from the tissue. The potential of leaching must be critically considered when using formalin-fixed biological tissue in trace metal analysis.     

Effect of fixation period on HER2/neu gene amplification detected by fluorescence in situ hybridization in invasive breast carcinoma.

This study investigated if formalin fixation duration affects HER2/neu gene amplification detection by fluorescence in situ hybridization (FISH) in breast cancer. Tumor tissues from 35 cases were divided into three groups and subjected to two formalin fixation protocols per group (12 hr, 27 hr in the first; 2 hr, 17.5 hr in the second; 28.5 hr, 541 hr in the third) before FISH analysis. There was no significant difference in FISH signal detection between the two different fixation protocols in the first two groups. In the third, no signal was detected in 4/6 cases fixed for an extended duration.     

Collagenase predigestion on paraffin sections enhances collagen immunohistochemical detection without distorting tissue morphology

Reliable immunohistochemical detection of collagen in formalin fixed, paraffin embedded tissues requires protease digestion. While these pan-proteases (pepsin, trypsin, protease K, etc.) enhance collagen detection, they also digest many other tissue proteins and produce poor cellular morphology and unrecognizable cellular structures. Balancing the conditions (protease type, concentration, incubation time and temperature) to digest some, but not all, proteins in a tissue section while optimizing collagen detection requires one to compromise improved collagen immunolabeling with adequate cellular morphology. Furthermore, optimal conditions for digesting tissue proteins to enhance collagen detection vary among tissue types and their fixation. Although brain is not typically subject to these deleterious consequences, structures such as epithelium, spermatids, stroma etc. and other tissues with complicated histology are profoundly affected. To resolve this technical dilemma, we discovered a novel use for collagenase to enhance collagen immunodetection without affecting the noncollagen proteins, thereby preserving tissue morphology. Collagenase, which is typically used in vitro for disassociation of cells, has never been used reliably on formalin fixed, paraffin embedded tissue sections. This new use of collagenase for immunohistochemistry promotes increased collagen immunolabeling, is easy to use, is versatile, and allows preservation of tissue structure that provides maximal and accurate histological information.     

Collagenase predigestion on paraffin sections enhances collagen immunohistochemical detection without distorting tissue morphology

Reliable immunohistochemical detection of collagen in formalin fixed, paraffin embedded tissues requires protease digestion. While these pan-proteases (pepsin, trypsin, protease K, etc.) enhance collagen detection, they also digest many other tissue proteins and produce poor cellular morphology and unrecognizable cellular structures. Balancing the conditions (protease type, concentration, incubation time and temperature) to digest some, but not all, proteins in a tissue section while optimizing collagen detection requires one to compromise improved collagen immunolabeling with adequate cellular morphology. Furthermore, optimal conditions for digesting tissue proteins to enhance collagen detection vary among tissue types and their fixation. Although brain is not typically subject to these deleterious consequences, structures such as epithelium, spermatids, stroma etc. and other tissues with complicated histology are profoundly affected. To resolve this technical dilemma, we discovered a novel use for collagenase to enhance collagen immunodetection without affecting the noncollagen proteins, thereby preserving tissue morphology. Collagenase, which is typically used in vitro for disassociation of cells, has never been used reliably on formalin fixed, paraffin embedded tissue sections. This new use of collagenase for immunohistochemistry promotes increased collagen immunolabeling, is easy to use, is versatile, and allows preservation of tissue structure that provides maximal and accurate histological information.     

Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction.

Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is increasingly being used to detect viral genomes and oncogene mutations. To determine the effect of fixation on the preservation of the nucleic acids, we fixed two randomly chosen fresh pathology specimens in formalin, B-5, Bouin's, Zenker's, ethanol, and Omnifix for 6, 24, 48, 72, and 168 hr (1 week), and then embedded the tissue in paraffin. Oligonucleotide primers specific for the cytoplasmic-beta-actin gene were chosen to span an intron such that amplification yielded a product of 250 BP for DNA and 154 BP for RNA. A single 6-microns section was cut from each paraffin block, deparaffinized, and then subjected to 30 rounds of amplification for either DNA or RNA. On amplifying DNA, consistent product was seen in the ethanol and Omnifix specimens up to 72 hr of fixation time, whereas variable product was seen with formalin or Zenker's fixation; all specimens fixed in Bouin's or B-5 were negative. On amplifying RNA, a product could be detected even after 1 week of fixation in ethanol or Omnifix, and after 48 hr in the formalin-fixed tissue. The Zenker's-fixed tissues gave variable results, and the Bouin's and B-5 tissues gave consistent results only after 6 hr of fixation. We therefore conclude that choice of fixative and fixation time are critical factors influencing the outcome of PCR amplification of nucleic acids from paraffin-embedded material.     

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。     

Studies on Bovine Mast Cells: I. The Effect of Formalin Fixation

Studies are reported on the effect of aqueous formalin fixation on the bovine mast cell. Paraffin sections of bovine skin were prepared from tissues fixed for 7 to 72 hr in 10% neutral formalin in saline, and from tissues which were treated with water for various times both before and after formalin fixation. Metachromatic halos, scattering of the granules, and fraying of the cell outline, were seen in the mast cells of tissues washed in water before fixation. Exposure to water after fixation did not produce these artifacts. The tendency towards orthochromatic staining, and the occurrence of perinuclear clear zones are probably effects of the formalin and not of overstaining or of exposure of the tissue to water. The majority of the metachromatic material in the bovine mast cell is water soluble, and may be removed by washing in water before fixation, whereas the granules are relatively resistant to water. The optimum time of formalin fixation of bovine skin to permit studies on the metachromatic material of the mast cells was 24 to 36 hr.     

The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.     

Effects of fixation for immunohistochemistry on tissue concentrations of substance P and somatostatin determined by radioimmunoassay

Concentrations of substance P and somatostatin were measured in preparations of the myenteric plexus (plus longitudinal muscle) of the guinea-pig ileum after fixation and processing for immunohistochemistry and compared with concentrations measured in fresh tissue. Two fixative solutions were used: (i) 4% formalin in phosphate buffer (0.1 M, pH 7.0); and (ii) a mixture of aqueous picric acid with 2% formalin in phosphate buffer (0.1 M, pH 7.0). Tissues were extracted in boiling aqueous acetic acid (2.0 M) either immediately after fixation and processing or after storage for up to four weeks in phosphate-buffered saline (PBS) with or without sodium azide. The concentrations of substance P and somatostatin in these extracts were measured by radioimmunoassay and compared to the concentrations in extracts of fresh tissue. The concentration of substance P in fixed tissue was the same as that found in fresh tissue, whereas the concentration of somatostatin in fixed tissue was half that found in fresh tissue (P<0.01). If the tissue was not subjected to the extensive washing for immunohistochemistry, somatostatin concentrations in fresh and fixed tissue were not significantly different. The concentration of substance P did not change on storage of the fixed tissue in PBS, either with or without sodium azide. The concentration of somatostatin decreased on storage of the fixed tissue in PBS over four weeks to 40% of its original value, but the presence of sodium azide maintained the concentration at 60% at four weeks. Neither fixative solution interfered with the radioimmunoassay except at very high concentrations. Fixation for 24h gave the highest estimates of each of the peptides. It is concluded that fixation can be a useful alternative to freezing for preservation of peptides in tissue for radioimmunoassay.     

Ultrasound-facilitated formalin fixation of biological specimens

Abstract

In a previous study, we showed that ultrasound can dramatically reduce the time required for tissue fixation in formalin. It generally is believed that ultrasound increases the speed of tissue fixation in two possible ways: 1) increasing the speed of penetration of fixative molecules into tissue samples and 2) increasing the speed of cross-linking reactions. We addressed here the second possible way by using protein solutions and cultured cells, which minimized the effects of the penetration factor. Proteins or cultured cells in solution were fixed with formalin with or without ultrasound irradiation. Fixed proteins and cell lysates then were separated by SDS-poly acrylamide gel electrophoresis and subjected to Western blotting to examine cross-linking formation in certain proteins. Unexpectedly, irradiation with ultrasound did not produce an observable difference in the rate of cross-linking in protein solutions. In similar experiments using cultured cells, however, we observed a significant reduction in recovery of certain proteins from cells fixed by formalin under the influence of ultrasound, which indicated that the ultrasound fixation procedure accelerated cross-linking formation within cells. Studies on protein and cell fixation without ultrasound showed that cross-linking formation was closely related to incubation temperature, which indicates that the heating function, which is inherently associated with ultrasound is another major factor in the ability of ultrasound to accelerate cross-linking.     

Tissue transglutaminase in the small intestine of the mouse as a marker for apoptotic cells. Colocalization with DNA fragmentation

Besides the morphological changes in cells undergoing apoptosis, such as chromatin condensation and cell shrinkage, histological demonstration of DNA fragmentation by in situ end labeling (ISEL) has been widely used for the demonstration of apoptotic cells in tissue sections. Although DNA fragmentation can be demonstrated in apoptotic cells and apoptotic bodies in most cases, there is no clear correlation of ISEL staining with apoptosis. It has often been demonstrated that, in many morphologically intact cells, nuclei with fragmented DNA can be found. Thus staining with ISEL for the detection of apoptosis is useful only in connection with other markers for apoptosis as, for example, characteristic morphological changes. Here we show that tissue transglutaminase protein is unequivocally expressed in apoptotic enterocytes as shown by DNA fragmentation and morphology. Tissue transglutaminase is not expressed in enterocytes with healthy morphology, although DNA fragmentation can be demonstrated in these cells. Thus the immunohistochemical demonstration of tissue transglutaminase may serve as a simple marker for apoptotic epithelial cells in tissue sections.     

Molecular mechanisms of antigen retrieval: antigen retrieval reverses steric interference caused by formalin-induced cross-links

Abstract

The overwhelming majority of antibodies useful for formalin fixed, paraffin embedded (FFPE) tissues require antigen retrieval to reverse the effect of formalin fixation and re-establish immunoreactivity. How this reversal happens is poorly understood. We developed a new experimental model for studying the mechanism of formalin fixation and antigen retrieval. Epitope mapping studies on nine antibodies useful for FFPE tissues revealed that each consisted of a contiguous stretch of amino acids in the native protein (linear epitope). Small peptides representing the epitopes of antibodies to human epidermal growth factor receptor type (HER2), estrogen, and progesterone receptors were attached covalently to glass microscope slides in a peptide array. Most peptides retained immunoreactivity after formalin fixation. Immunoreactivity was completely abrogated for all peptides, however, if an irrelevant large protein was present during formalin-induced cross-linking. We hypothesize that cross-linking the irrelevant protein to the peptide epitopes sterically blocked antibodies from binding. Antigen retrieval dissociates irrelevant proteins and restores immunoreactivity. Because the epitopes for clinical antibodies require only primary protein structure, the fact that antigen retrieval probably denatures the secondary and tertiary structure of the protein is irrelevant. The same mechanism may occur in tissue samples subjected to formalin fixation and antigen retrieval.     

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.     

Histochemical study of apoptotic epithelial cells depending on testosterone in primary cultured rat prostatic tissues

To clarify whether apoptosis can be induced in cultured rat prostatic epithelial cells, they were investigated at various time points, depending on different concentrations of testosterone. Ventral lobes of rat prostates were cultured as small pieces of tissues up to 14 days. They were examined by anti-Fas antibody immunostaining and also compared to findings revealed by in situ end-labelling (ISEL) technique. To clarify apoptotic nuclei at high resolution, the quick-freezing and deep-etching (QF-DE) method was also used, as reported before. The localization and appearance of Fas-positive cells were detected more widely and earlier than those of ISEL-positive cells, but both label-positive localizations were closely related to each other. In addition, they were detected more often in epithelial cells cultured with low testosterone concentrations. By the QF-DE method, chromatin fibers were found to be broken in spotty parts of apoptotic nuclei. We could control the concentration of testosterone in culture medium and detect the appearance of Fas antigen in cultured prostatic epithelial cells, followed by apoptotic changes. So, Fas and Fas-ligand system is one candidate for apoptosis in the prostate glands, depending on removal of hormonal testosterone.     

Detection and localization of HIV-1 DNA in renal tissues by in situ polymerase chain reaction

The localization of HIV-1 DNA in renal tissues is critically important for understanding pathogenesis of HIV-associated nephropathy (HIVAN), but the clarification has been technically challenging. We applied in situ polymerase chain reaction (IS-PCR) to human renal tissues to demonstrate viral entry into the renal epithelial cells in vivo. To test the specificity of this method and to determine the cell types infected, we used IS-PCR followed by in situ hybridization (ISH) and IS-PCR followed by immunohistochemistry and histochemical counterstains. Brief 2 hour fixation in 4% paraformaldehyde had 92.9% sensitivity and 100% specificity for detection of viral DNA in renal biopsies of HIVAN patients, compared to 70.8% sensitivity and 66.7% specificity in renal biopsies fixed overnight in 10% formalin. Under optimized conditions, the only signals detectable in HIV-1 seronegative cases were false positives attributable to renal tubular apoptosis. In HIVAN cases, positive signal was observed in podocytes, parietal cells, renal tubular cells, and interstitial leukocytes. Immunohistochemical co-labeling for pan-T cell and macrophage markers revealed that the interstitial leukocytes with positivity for HIV-1 DNA included both T cells and macrophages. Application of ISH after IS-PCR showed the same distribution of signal as observed using IS-PCR alone, confirming the specificity of the technique. IS-PCR is a powerful technique to detect viral DNA in human tissue sections, but requires proper use of negative controls to set optimal fixation, protein digestion, and amplification conditions.     

Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step

Summary We examined the application of BrdUrd immunohistochemistry to detect S-phase cells in undecalcified bone and cartilage from the growing rat embedded in Spurr's resin. The effect of fixation on the procedure was studied, and the validity of the technique examined by a comparative study with tritiated thymidine ([³H]-TdR) autoradiography. The use of sodium-ethoxide to remove plastic from tissue sections prior to immunohistochemistry resulted in the production of sufficient ssDNA to make a separate DNA denaturation step unnecessary, thus sparing sections from potentially destructive treatment and shortening the immunohistochemical procedure. Fixation in formalin or Bouin's fluid gave the most satisfactory results. The distribution of BrdUrd labeled cells was restricted to the sites of cell proliferation in growing long bones. Combined studies with BrdUrd immunohistochemistry and [³H]-TdR autoradiography showed that the majority of BrdUrd labeled cells had also incorporated [³H]-TdR, thus attesting to the validity of the technique. This novel approach is suitable for the study of undecalcified hard tissues as well as soft tissues.     

Alcoholic fixation over formalin fixation: A new,safer option for morphologic and molecular analysis of tissues

Formalin is a widely used fixative but there is potential public health risks to exposure. Besides, alcoholic fixation is advantageous over formalin fixation because of faster fixation, optimal preservation and safer workplace environment. Following fixation by EMA and 10% neutral buffered formalin (NBF), we analyzed the tissue morphology, antigenic stability, DNA and RNA quantity with quality (OD value). The findings of EMA fixing on both the tissue morphology and molecular characterization, were satisfactory. Specially, EMA was faster in penetration of tissues than NBF, fixed ideally as early as 8 h of fixation whereas improper fixation was evident for NBF. In Hematoxylin and Eosin (H & E) staining, better cellular details with stronger affinity for staining were observed. In immunohistochemistry, better antigenic stability was reported for EMA-fixed tissues. The nucleic acid analysis revealed that total genomic DNA and RNA yield from EMA fixed tissues were significantly higher (P < 0.05) with superior quality than NBF fixed tissues. Our results suggest that EMA could be a potential alternative to NBF for fixation and preservation of tissues. These data provide new insights into an option for a safer working environment to support study and research.     

The Use of Mouse Models of Breast Cancer and Quantitative Image Analysis to Evaluate Hormone Receptor Antigenicity after Microwave-assisted Formalin Fixation

Microwave methods of fixation can dramatically shorten fixation times while preserving tissue structure; however, it remains unclear if adequate tissue antigenicity is preserved. To assess and validate antigenicity, robust quantitative methods and animal disease models are needed. We used two mouse mammary models of human breast cancer to evaluate microwave-assisted and standard 24-hr formalin fixation. The mouse models expressed four antigens prognostic for breast cancer outcome: estrogen receptor, progesterone receptor, Ki67, and human epidermal growth factor receptor 2. Using pathologist evaluation and novel methods of quantitative image analysis, we measured and compared the quality of antigen preservation, percentage of positive cells, and line plots of cell intensity. Visual evaluations by pathologists established that the amounts and patterns of staining were similar in tissues fixed by the different methods. The results of the quantitative image analysis provided a fine-grained evaluation, demonstrating that tissue antigenicity is preserved in tissues fixed using microwave methods. Evaluation of the results demonstrated that a 1-hr, 150-W fixation is better than a 45-min, 150-W fixation followed by a 15-min, 650-W fixation. The results demonstrated that microwave-assisted formalin fixation can standardize fixation times to 1 hr and produce immunohistochemistry that is in every way commensurate with longer conventional fixation methods.