Ionic liquids in embalming and tissue preservation. Can traditional formalin-fixation be replaced safely?

Ionic liquids (ILs) can be used for embalming and tissue preservation. ILs does not cause tissue damage and the tissue colour remains unaltered after treatment. Microscopical morphology of tissues fixed in ILs is of better quality than that of tissues fixed in formalin. Tissue preservation depends on the type of ILs. Best results were obtained with 1-methyl-3-octyloxymethylimidazolium tetrafluoroborate, the density of which resembles that of water. The salt is nonvaporous and when used as a formalin substitute, it eliminates health hazards in the pathological laboratory.     

Inadequate formalin fixation decreases reliability of p27 immunohistochemical staining: probing optimal fixation time using high-density tissue microarrays

Immunohistochemical analysis of molecular targets in clinical tissues is increasingly becoming central to our ability to render diagnoses, to predict prognosis, to select patients for appropriate therapies, and to provide surrogate end points for therapeutic monitoring. For example, reduction of immunohistochemical staining for the cyclin-dependent kinase inhibitor p27(Kip1) has been proposed as a potential prognostic biomarker in prostate, breast, and gastrointestinal tumors. We observed that with our standard formalin fixation in rapidly processed (same-day) radical prostatectomy specimens, there is often a gradient of p27(Kip1) staining in normal prostate epithelium, with more staining near the periphery and less staining toward the center of the sample. This raised the hypothesis that the reliability of staining for p27(Kip1) is decreased in inadequately fixed tissues. The implications of this, if true, are that many studies using p27(Kip1) for prognostic purposes may be subject to unpredictable artifacts, and hence unreliable results, if the fixation of the specimens is not well controlled. The objectives of the present study were (1) to formally test the hypothesis that inadequate fixation time is responsible for apparent loss of p27(Kip1) nuclear staining and (2) to test a recently proposed method for improving the uniformity of immmunohistochemical staining using formalin injection. Prostate tissue sections from radical prostatectomy specimens were either processed immediately (zero time fixation) or fixed for 1, 2, 3, or 8 days in 10% neutral buffered formalin before processing into paraffin. To assure identical antigen retrieval and immunohistochemical staining conditions for specimens fixed for different lengths of time, 2 high-density tissue microarrays (TMAs), containing 564 tissue samples (0.6 mm in diameter) were constructed. Based on an estimate of the percentage of nuclei in normal prostatic epithelial secretory cells with strong staining, quality of p27(Kip1) staining was graded in a blinded fashion with respect to fixation time. There was a significant increase in the percentage of cores that were scored as "strong" as fixation time increased from 0 (same-day processing) to 1 or more days (P <.0001). Interestingly, even at 8 days of fixation, there was excellent staining that was superior to the same-day processing. Based on these results, we conclude the following: (1) for large clinical specimens that have been fixed briefly to decrease diagnostic turn-around time, the reliability of interpretation of immunohistochemical staining may be quite limited; (2) for p27(Kip1), decreased antigen staining as a result of the widely held concept of "overfixation" is much less of a problem than "underfixation"; (3) formalin injection produces a marked improvement in staining for several markers, including p27(Kip1); and (4) high-density TMAs, which assure identical test conditions, provide an excellent platform on which to evaluate the effects of tissue fixation on immunohistochemical staining.     

Use of freeze-drying technology for the study of renal biopsies

Adequate handling of renal biopsies requires processing for light and electron microscopy, as well as for immunohistochemical study. Problems of adequate sampling are frequently encountered, and tissue size can be insufficient, since morphologic examination requires chemical fixation, while immunofluorescence study is currently performed on snap-frozen, cryostat-cut tissue. The application of freeze-drying technology on 20 renal needle biopsies has been investigated to assess the feasibility and reliability of the technique as a routine diagnostic tool in renal pathology. Morphologic as well as immunohistochemical studies were performed on freeze-dried, paraffin-embedded specimens, including immunofluorescence and PAP techniques to detect immunoglobulins, complement fractions, fibrinogen, collagen types, and laminin. Our results showed good preservation of tissue morphology, similar to standard formalin fixation. Moreover, absence of diffusion artifacts and intensity of immunohistochemical reactions were comparable to what is obtained with cryostat sections. We therefore suggest that freeze-drying before paraffin embedding is, at least for diagnostic purposes, preferable to formalin fixation; unfixed, cryostat-cut sections can still be informative and should be used whenever tissue is available in selected cases, and/or in experimental work.     

Evaluation of Streck tissue fixative, a nonformalin fixative for preservation of stool samples and subsequent parasitologic examination

We undertook a study to evaluate Streck tissue fixative (STF) as a substitute for formalin and polyvinyl alcohol (PVA) in fecal preservation. A comparison of formalin, PVA, (mercuric chloride based), and STF was done by aliquoting fecal samples into each fixative. Stool specimens were collected in Haiti, and parasites included Cyclospora cayetanensis, Giardia intestinalis, Entamoeba coli, Iodamoeba butschlii, Endolimax nana, Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis, and Necator americanus. Preserved stools were examined at various predetermined times (1 week, 1 month, and 3 months) to establish the quality of the initial preservation as well as the suitability of the fixative for long-term storage. At each time point, stool samples in fixatives were examined microscopically as follows: (i) in wet mounts (with bright-field and epifluorescence microscopy), (ii) in modified acid-fast-, trichrome-, and safranin-stained smears, and (iii) with two commercial test kits. At the time points examined, morphologic features remained comparable for samples fixed with 10% formalin and STF. For comparisons of STF- and 10% formalin-fixed samples, specific findings showed that Cyclospora oocysts retained full fluorescence, modified acid-fast- and safranin-stained smears of Cryptosporidium and Cyclospora oocysts were equal in staining quality, and results were comparable in the immunofluorescence assay and enzyme immunoassay commercial kits. Stool fixed in STF and stained with trichrome showed less-than-acceptable staining quality compared with stool fixed in PVA. STF provides an excellent substitute for formalin as a fixative in routine examination of stool samples for parasites. However, modifications to the trichrome staining procedures will be necessary to improve the staining quality for protozoal cysts fixed in STF to a level comparable to that with PVA.     

Ultrasound-accelerated formalin fixation of tissue improves morphology, antigen and mRNA preservation.

Formalin fixation and paraffin embedding are conventional tissue preservation and processing methods used for histologic diagnosis in over 90% of cases. However, formalin fixation has three disadvantages: (1) slow fixation (16-24 h) hinders intraoperative decision making, (2) slow quenching of enzymatic activity causes RNA degradation, and (3) extensive molecule modification affects protein antigenicity. Applying high-frequency, high-intensity ultrasound to the formalin fixative cuts fixation time to 5-15 min. Fixation of various tissues such as lymph node, brain, breast, and prostate suggests that, compared to the conventional method, implementation of ultrasound retains superior and more uniform tissue morphology preservation. Less protein antigenicity is altered so that rapid immunohistochemical reactions occur with higher sensitivity and intensity, reducing the need for antigen retrieval pretreatment. Better RNA preservation results in stronger signals in in situ hybridization and longer RNA fragments extracted from fixed tissues, probably due to rapid inhibition of endogenous RNase activity. Molecules extracted from ultrasound-fixed tissues are of greater integrity and quantity compared to conventionally fixed tissues, and thus better support downstream molecular analyses. Overall, ultrasound-facilitated tissue preservation can provide rapid and improved morphological and molecular preservation to better accommodate both traditional and molecular diagnoses.     

DEVELOPMENT OF AN OPTIMAL PROTOCOL FOR ANTIGEN RETRIEVAL: A ‘TEST BATTERY’ APPROACH EXEMPLIFIED WITH REFERENCE TO THE STAINING OF RETINOBLASTOMA PROTEIN (pRB) IN FORMALIN-FIXED PARAFFIN SECTIONS

The retinoblastoma (RB) gene, which encodes the nuclear RB protein (pRB), is believed to be involved in cell cycle control and cell differentiation. Studies have demonstrated that loss of RB function may play a role in tumour formation and progression of a variety of human tumours, such as bladder, lung, breast, and prostate cancers. The immunohistochemical detection of pRB expression in formalin–paraffin sections of human cancer has potential advantages of convenience, economy, and compatibility with routine surgical pathology practice. In practice, however, results using pRB antibodies on routinely processed, paraffin-embedded tissue have been inconsistent. In this study, the antigen retrieval (AR) method has been applied to the immunohistochemical detection of pRB in paraffin-embedded tissues and a ‘test battery’ approach has been developed to identify the principal variables that result in the optimal AR protocol. This approach includes the use of buffered solutions at pH 1, 6, and 10 with three different heating conditions (temperatures 120°C, 100°C, and 90°C). In the example described here with antibody RB-WL-1, the low pH solution with the microwave heating at 100°C proved most effective. Both fresh and routinely processed formalin–paraffin tissues of normal and bladder carcinoma were used for a comparison of the pRB immunostaining. The AR method was evaluated by comparing the immunohistochemical staining result on routinely processed formalin–paraffin sections with frozen sections of the same tumour. A consistent intensity of immunohistochemical staining for pRB was achieved using the identified optimal AR protocol on formalin–paraffin sections. All slides showed positive staining of pRB in normal mesenchymal and epithelial tissues. The pattern of pRB localization and intensity of staining was similar to that obtained in frozen sections, though the intensity obtained by AR treatment on paraffin sections was slightly to moderately stronger than that obtained in frozen sections. Once the protocol was identified, it was tested using routinely processed paraffin tissue sections of 245 cases of bladder carcinoma, with consistent pRB immunostaining results. The protocol described is simple to perform and gives reproducible results for evaluation of pRB expression by immunohistochemistry.     

Comparison of formalin- and acetone-fixation for immunohistochemical detection of carcinoembryonic antigen (CEA) and keratin

The purpose of the present study was to compare the relative merits of cold acetone and buffered formalin as fixatives for the detection of carcinoembryonic antigen and keratin in permanently embedded tissues using a peroxidase-antiperoxidase (PAP) immunohistochemical procedure. The effect of treatment with the proteolytic enzyme pronase also was examined in the formalin-fixed tissues. Cold acetone was found to be superior to formalin for the preservation of CEA and keratin antigenic activities in a variety of benign and malignant tissues. Pronase treatment markedly increased the staining intensities of both antigens in formalin-fixed tissues. For many tissues, however, superior results were obtained using the cold acetone method, and this technic is recommended for the optimum retention of antigenic activity in permanently embedded tissues.     

Restoration of antigenicity of tissue antigens, cell-bound immunoglobulins and immune deposits in paraffin-embedded tissue. The influence of fixation and proteolytic enzymatic digestion

The possibility of preservation and restoration of antigenicity of some antigens in paraffin-embedded tissue was evaluated by direct immunofluorescent technique on deparaffinized sections. Fixation with 96% ethanol-1% acetic acid, 10% neutral buffered formalin and p-formaldehyde was useful for the preservation of tissue antigens and immune deposits, whose antigenicity could be easily restored by trypsin digestion. Neutral buffered formalin was also a satisfactory fixative in immunofluorescent staining on lymphocyte/plasma cell-bound immunoglobulins. Fixation with alcohol-Bouin's fluid showed contrast results; feasible for staining of cell-bound immunoglobulins, but poor for that of glomerular immune deposits. After papain digestion, BSA and lysozyme, antigens of immune complexes, were easily detected in experimental chronic serum sickness glomerulonephritis. Pepsin was more efficient than trypsin in restoring the antigenicity of renal tissue antigens such as fibronectin and polyantigenic basement membrane, but the brush border antigen of the proximal renal tubules was frail to the pepsin digestion. In general, the enzymatic digestion time necessary for the restoration of antigenicity was in parallel with fixation time. Results obtained have shown that deparaffinized sections could be used as satisfactory substrate for immunohistochemistry when proper fixation and efficient proteolytic enzymatic pretreatments were performed.     

Use of immunocytochemistry and biotinylated in situ hybridisation for detecting measles virus in central nervous system tissue.

Optimised immunocytochemical (ICC) and in situ hybridisation (ISH) protocols for long term, formalin fixed, central nervous system tissue infected with measles virus were developed. The effectiveness of 10 proteases for the enzymatic unmasking of formalin fixed antigen and nucleic acid was investigated. Protease VIII gave maximal signal generation with optimal tissue preservation and no background staining for both techniques. The use of a microwave oven as an additional pre-hybridisation step for RNA-RNA in situ hybridisation produced a significant increase in the number of cells labelled for genomic RNA. The ability to show the presence of antigen and nucleic acid in long term, formalin fixed tissue facilitates the use of stored necropsy material available in pathology departments for ICC and ISH investigations.     

Assessment of oestrogen receptor content of breast carcinoma by immunohistochemical techniques on fixed and frozen tissue and by biochemical ligand binding assay.

The oestrogen receptor content of 61 breast carcinomas was assessed by biochemical ligand binding assay and three immunohistochemical techniques--a frozen section method (Abbott ER-ICA) and on paraffin wax sections after fixation by two methods. The two fixatives used were Carson's buffered formalin and methacarn, and a DNAse pretreatment of sections was used. Overall agreement for the immunohistochemical methods with the ligand binding technique were 95%, 85%, and 86% for the frozen, formalin, and methacarn methods, respectively. A semiquantitative staining score was performed and all three methods gave significant correlations of staining scores with biochemical ligand binding values. The frozen section method was best (r = 0.88) with the fixed tissue methods yielding poorer correlation coefficients. Several factors affected staining, including the nature of the fixative and variable activity of DNAse. It is concluded that immunohistochemical assessment of oestrogen receptor content on fixed tissue provides acceptable qualitative information but that standardisation of protocols for tissue processing will be necessary for optimal utility and especially for quantitative assessments.     

Restoration Of Antigenicity Of Tissue Antigens,Cell-Bound Immunoglobulins And Immune Deposits In Paraffin-Embedded Tissue The Influence Of Fixation And Proteolytic Enzymatic Digestion

The possibility of preservation and restoration of antigenicity of some antigens in paraffin-embedded tissue was evaluated by direct immunofluorescent technique on deparaffinized sections. Fixation with 96% ethanol-1% acetic acid, 10% neutral buffered formalin and p-formaldehyde was useful for the preservation of tissue antigens and immune deposits, whose antigenicity could be easily restored by trypsin digestion. Neutral buffered formalin was also a satisfactory fixative in immunofluorescent staining on lymphocyte/plasma cell-bound immunoglobulins. Fixation with alcohol-Bouin's fluid showed contrast results; feasible for staining of cell-bound immunoglobulins, but poor for that of glomerular immune deposits. After papain digestion, BSA and lysozyme, antigens of immune complexes, were easily detected in experimental chronic serum sickness glomerulonephritis. Pepsin was more efficient than trypsin in restoring the antigenicity of renal tissue antigens such as fibronectin and polyantigenic basement membrane, but the brush border antigen of the proximal renal tubules was frail to the pepsin digestion. In general, the enzymatic digestion time necessary for the restoration of antigenicity was in parallel with fixation time. Results obtained have shown that deparaffinized sections could be used as satisfactory substrate for immunohistochemistry when proper fixation and efficient proteolytic enzymatic pretreatments were performed. ACTA PATHOL JPN. 34: 563–574, 1984.     

The utility of vapor fixation for cell block preparation in fine needle aspiration of malignant lesions of lymph nodes

Cell blocks overcome certain limitations of fine needle aspiration cytology (FNAC), such as study of tissue architecture and application of immunohistochemistry (IHC). The main disadvantage of cell block by conventional method is cell loss. This study compares formaldehyde vapor-fixed cell blocks with conventional formalin-fixed blocks obtained from aspirates of malignant lesions of lymph nodes. Seventy-nine patients with suspected primary or metastatic malignancy in lymph nodes were studied. Cytologic smears were prepared in all the cases. Conventional formalin-fixed cell blocks were made in 34 cases, vapor-fixed cell blocks in 34 cases, and in the remaining 11 cases, both methods were employed. Cell block sections were compared for cellularity, fixation artifacts, morphologic details, and crispness of immunohistochemical staining using defined subjective criteria. Cellular yield was slightly less with vapor-fixed cell blocks in comparison with conventional formalin-fixed cell blocks. Staining artifacts were more frequent with vapor fixation (33.3%) as compared to conventional formalin fixation (20%). Also, vapor fixation required more time (6 h) as compared to conventional formalin fixation (1 h). There was no difference in the two techniques with respect to immunohistochemical staining. Cell blocks prepared by vapor fixation are comparable to conventional formalin-fixed cell blocks.     

Systematic comparison of tissue fixation with alternative fixatives to conventional tissue fixation with buffered formalin in a xenograft-based model

In our study we systematically compared the alternative fixatives acidified formal alcohol (AFA), PAXgene?, HOPE?, and combinations of AFA or formalin with ultrasound treatment to standard (buffered) formalin fixation. We examined general morphology and detectability of protein structures by immunohistochemistry of the membrane receptors epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF-1R), and phosphorylated human epidermal growth factor receptor 2 (phospho-HER2). In order to allow for stringent comparability of different fixation techniques, we used matched mouse xenograft tumor samples from three different human cancer cell lines (colon, ovarian, and non-small cell lung cancer), either fixed conventionally with formalin or an alternative fixative. Tissue morphology after fixation with AFA and PAXgene? was comparable to formalin-fixed paraffin-embedded tissue (FFPET) morphology. Ultrasound fixations resulted in slightly inferior morphology and HOPE? fixation preserved morphology only poorly compared to FFPET in this system. None of the tested alternative fixatives enabled immunohistochemical detectability of all three targets in the same manner as FFPET. Pronounced staining was possible for EGFR and IGF-1R with all alternative fixatives but HOPE?, and phospho-HER2 staining was only noteworthy with formalin-ultrasound-fixed tissue. Therefore, the use of alternative fixatives comes with the need for careful validation of obtained IHC results individually for each target.     

Tissue fixation effects on immunohistochemical staining of caspase-3 in brain tissue.

Fixation methods for tissue often vary amongst clinical and research laboratories. To evaluate the effects of fixation method on studies of brain tissue, we examined immunohistochemical outcomes amongst 2 fixatives, 4 caspase-3 antibodies, and 2 species (human infants and piglets). Fixatives were 10% neutral buffered formalin (NBF) or 10% NBF and glacial acetic acid (FAA). Antibodies for caspase-3 were commercially obtained and included 2 for active caspase-3, and 2 for procaspase-3 (CASP3 and CPP32). Immunohistochemical staining of caspase-3 varied with fixation method, with the greatest effect of fixation method observed for the active caspase-3 antibodies and this effect was present in both species. In NBF-fixed tissue, active caspase-3 immunoreactivity was only visible microscopically, and was specific to neuronal cell bodies. In FAA-fixed tissue, active caspase-3 immunoreactivity was visible macroscopically, and predominantly present in fiber tracts and fasciculi compared with neuronal bodies. Fixation and species differences were also identified for the procaspase-3 antibodies, CASP3 and CPP32, where FAA-fixed pig tissue showed abundant staining of blood vessels that were not observed in the NBF-fixed pig tissue or in the human tissue. This study characterizes differences in immunohistochemical outcomes using commercially available antibodies for caspase-3, according to tissue fixation method and species.     

The effects of glyoxal fixation on the histological evaluation of breast specimens

Glyoxal (GL), a non-formalin-containing aldehyde tissue fixative, is advocated as a superior fixative that is environmentally safe and lacks the purported carcinogenic health hazards associated with formalin use. In addition, it is advertised as requiring no antigen retrieval before immunohistochemical staining. We compared GL fixation to standard formalin fixation of breast specimens removed for microcalcifications or breast tumors. Although the hematoxylin and eosin morphology of GL-fixed and formalin-fixed tissues was equivalent, detection of microcalcifications in GL-fixed breast specimens was hampered by loss of basophilia, likely due to increased calcium solubility in glyoxal. Moreover, estrogen receptor detection in GL-fixed specimens was diminished compared to formalin and did require antigen retrieval.     

Demonstration of oestrogen and progesterone receptors in freeze-dried, paraffin-embedded sections of breast cancer

Immunohistochemical evaluation of oestrogen and progesterone receptors is of importance in evaluating human breast tumours. Staining techniques can be performed on snap-frozen, cryostat-cut tissues or, as recently reported, on formalin-fixed, paraffin-embedded tissues. These methods are, however, limited by several drawbacks, including difficulties in retrospective studies and in storage of the material, and the relatively high frequency of false negative results for chemically fixed specimens. We therefore investigated the application of freeze-drying technology to assess the feasibility and reliability of this technique as an alternative method for diagnostic breast pathology. Morphological and immunohistochemical studies were performed on snap-frozen, freeze-dried and paraffin-embedded tissue obtained from 16 cases of benign and malignant breast neoplasms. Our results showed good preservation of tissue morphology, similar to standard formalin fixation, and excellent preservation of antigenic reactivity of nuclear receptors, comparable to that obtained with cryostat sections. We therefore suggest that freeze drying and paraffin embedding of frozen tissue blocks is equivalent or even preferable to formalin fixation for the demonstration of oestrogen and progesterone receptors, at least in the case of small tumours.     

Microwave irradiation as a form of fixation for light and electron microscopy

Microwave irradiation was used to fix a wide range of surgical and autopsy specimens as well as tissue from freshly killed rats. Although there appeared to be varying optimum temperatures of fixation for different tissues, from a practical standpoint, the heating to 58 degrees C of tissues submerged in normal saline resulted in fixation of a quality comparable with that produced by conventional fixation to 10 per cent formalin. Microwave irradiation applied to tissues which had up to 1 h prior immersion in 10 per cent formalin also produced excellent preservation of cytomorphology, the time required for microwave fixation being no more than 150 s. This form of rapid heat fixation had no deleterious effects on special stains, sectioned as well as control fixed blocks and produced less shrinkage artefact than conventional formalin fixation. Immunocytochemical staining for more stable cytoplasmic antigens revealed no significant difference between microwave fixation compared with formalin fixation. The more labile membrane associated antigens of lymphocytes, however, were not preserved by either method of fixation. Microwave fixation was also found to be applicable to electron microscopy. Tissue samples immersed in 2.5 per cent glutaraldehyde and fixed in 90 s by irradiation to 50 degrees C showed excellent preservation of ultrastructural morphology.     

Effects of isopropanol storage time on histochemical and immunohistochemical stains in lung tissue

Abstract

Objectives: Prolonged storage of tissues in formalin is known to result in progressively increasing cross-linking of proteins and subsequent difficulties in histochemical and immunohistochemical staining. One solution to this problem is to fix the tissues in formalin and then store them in alcohol. Our institution uses isopropanol for this purpose. However, little is known on how isopropanol storage may affect the quality of subsequent staining of sections taken from these tissues.

Methods: To test this hypothesis, lung tissue samples from a transplant donor lung were fixed for 24 hours in 10% neutral buffered formalin and then stored in 70% isopropanol at 4°C for 0 days, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, and 24 weeks. All lung tissue samples were stained for hematoxylin and eosin, Masson’s trichrome, periodic acid Schiff, CD31, CD68, and smooth muscle actin. Staining intensities were scored (1?=?weak; 2?=?moderate; and 3?=?intense) and the inter-observer variability was evaluated.

Results and discussion: There were no significant differences in the staining intensities of hematoxylin and eosin, Masson’s trichrome, periodic acid Schiff, CD31, CD68, and smooth muscle actin in the lung tissue samples with increasing storage time in isopropanol. Thus, the tissues that are stored in isopropanol at 4°C can yield good quality staining for research and clinical investigations.     

Formaldehyde substitute fixatives. Analysis of macroscopy, morphologic analysis, and immunohistochemical analysis

Because formaldehyde is toxic and creates cross-links that may hinder immunohistochemical studies, we tested 3 new cross-linking (F-Solv [Adamas, Rhenen, the Netherlands]) and non-cross-linking (FineFIX [Milestone, Bergamo, Italy] and RCL2 [Alphelys, Plaisir, France]) alcohol-based fixatives for routine staining in comparison with neutral buffered formalin (NBF) as the "gold standard." Fresh tissue samples were divided into 4 equal pieces and fixed in all fixatives for varying times. After paraffin embedding, H&E staining, 7 common histochemical stains, and 9 common immunohistochemical stains were performed. RCL2 fixation resulted in soft and slippery tissue, causing sectioning difficulties. F-Solv and FineFIX led to partial tissue disintegration during fixation. F-Solv performed morphologically similar to NBF but needed considerable protocol adjustments before being applicable in daily histologic and immunohistochemical practice. FineFIX did not necessitate major protocol changes but caused shrinkage artifacts, degranulation, and lysis of RBCs. RCL2 generated morphologically overall good results without major protocol changes but caused pigment deposition, degranulation, and RBC lysis. The alcohol-based fixatives had positive and negative attributes and environmental drawbacks, and none was overall comparable to NBF with regard to macroscopy, morphologic evaluation, and immunohistochemical studies.     

Immunohistochemistry of neurone specific enolase with gamma subunit specific anti-peptide monoclonal antibodies.

AIMS--To investigate the application in immunohistochemistry of gamma-subunit specific anti-peptide monoclonal antibodies to human neurone specific enolase (NSE); and to determine their reactivity with formalin fixed, wax embedded sections of normal tissue and neuroendocrine tumours. METHODS--Immunohistochemical staining was performed on sections of formalin fixed, wax embedded tissue with two monoclonal antibodies (NSE-P1 and NSE-P2) raised against different synthetic peptides specific for the gamma subunit of human enolase (neurone specific enolase). RESULTS--Both antibodies gave strong immunostaining in normal tissues and cells known to contain NSE. There was no immunoreactivity in tissues containing either the alpha alpha or beta beta isozymes of enolase. The reactivity of the antibodies with a range of neuroendocrine tumours was also studied and both antibodies gave strong immunostaining of tumour cells in the different tumours. CONCLUSIONS--The use of synthetic peptides from defined regions of a molecule as immunogenes provides antibodies of high specificity. These monoclonal antibodies to NSE are ideally suited for immunohistochemical studies and they should be particularly useful in histopathology as they react with epitopes which are resistant to formalin fixation and wax embedding.