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.     

KP1 Immunohistochemical Demonstration of Macrophages in Colorectal Tissues: Effects of Different Fixatives and Different Fixation Times

Abstract

The effects of 10 fixatives and formalin fixation time were assessed on the immunoreactivity of KP1 (CD68 antigens) in colorectal tissues in paraffin embedded sections. A panel of fixatives including B5, periodate-lysine-paraformaldehydedichromate, periodate-lysine-paraformaldehyde, 4% paraformaldehyde (PF), zinc formalin, formol dichromate, 10% neutral buffered formalin (NBF), Bouin fluid, Carnoy fluid, and acetone were used to determine an optimal fixative. Another set of colorectal tissues were fixed in 10% NBF progressively at intervals of 1,2,4, and 20 hr; 2,7, and 14 days; and 4 wk to investigate the formalin-induced detrimental effect on CD68 antigens of macrophages.

The best result was found after fixation in B5. Short fixation in NBF for 4 hr with trypsinization also gave satisfactory results. There was a distinct decrease in staining intensity after 20 hr exposure to NBF, and the reactivities were totally abolished after 2 days. Trypsinization did enhance the staining intensity significantly with no discrimination of fixatives. (The J Histotechnol 17:329, 1994)     

The effect of a series of fixatives on the AgNOR technique

With increasing interest being shown in nucleolar organizer regions (NORs) in pathology, it was considered of great importance to evaluate the effect of some of the more commonly used and more specialized fixatives on the demonstration of these moieties. NORs can be demonstrated in paraffin sections by a silver technique (AgNOR method) which was developed from a method used by cytogeneticists for the demonstration of NORs in chromosome spreads. The degree of staining is dependent on the fixation regime employed and results may vary greatly from one fixative to another. The fixative schedules and post-treatments used in this study were based on standard sequences from the literature. We have shown that, in general, alcohol-based fixatives give optimal results, Carnoy's fluid being especially recommended. Mercurial and dichromate-containing fixatives were found to have highly detrimental effects on NOR staining. 'Routine' 10 per cent formol saline fixation gave adequate results whereas 10 per cent neutral buffered formalin gave optimal staining, similar to alcohol-based fixation.     

The effect of fixatives and paraffin embedding on the histochemical reactivity of a monoclonal antibody against human type IV collagen (JK-199)

A new monoclonal antibody (JK-199) was found to react with basement membranes on paraffin-embedded tissue sections without prior enzyme digestion. JK-199 was shown to react with isolated type IV collagen treated by any of four different fixatives--PLP, 4% formalin, modified Zamboni's (0.2% picric acid, 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) or Bouin's--applied for 6 h at room temperature and incubated at 60 degrees C for 30 min to simulate routine tissue processing. None of the fixatives was able to alter the reactivity of JK-199 with isolated type IV collagen. In the human placenta, specific and intense staining of basement membranes was demonstrated on paraffin sections fixed with any of the four fixatives. In human skin, basement membranes were fully demonstrated on paraffin sections fixed by PLP fixative or by 4% formalin, but only partially on sections fixed by picric acid-containing fixatives. Optimal results, i.e., with the least non-specific or incomplete staining, were obtained on PLP-fixed paraffin-embedded tissues. In PLP-fixed paraffin sections of the kidney, skeletal muscle, and small intestine, all basement membranes were stained intensely by this antibody (JK-199) at the expected locations. The results indicate that JK-199 may be widely applicable for the analysis of basement membrane kinetics, including developmental processes or pathological conditions.     

Immunohistochemical detection of fibronectin using different fixatives in paraffin embedded sections

Different fixatives and immunohistochemical methods were tested for detection of fibronectin in various paraffin embedded tissues: rat kidney, spleen, gastro-intestinal tract, muscle, normal and fibrotic liver and human skin. Using cryostat sections, localisation with immunofluorescence and peroxidase technics comparable to those obtained in unfixed tissue sections, could be obtained with the following fixatives: 10% formalin in PBS containing 4% sucrose; 96% ethanol; 96% ethanol + 1% acetic acid; a series of ethanol solutions of increasing strength: 70-80-96%. These fixatives also proved to be the best for paraffin embedding. Without enzyme digestion, however, satisfactory results could not be obtained with either indirect peroxidase or immunofluorescence methods in paraffin embedded tissues. Following digestions with the enzymes at the concentrations described in the literature, the alteration of tissues made the morphological localization of fibronectin difficult. The self-sandwich peroxidase method following a gentle pepsin digestion gave results closest to those of unfixed cryostat sections; however a slight increase in background staining was observed but without interfering with the evaluation of results.     

Effect fixation on T and B lymphocyte surface membrane antigen demonstration in paraffin processed tissue

The identification of lymphoid surface membrane antigens in tissue sections using immunohistochemical techniques is becoming increasingly important for the diagnosis and classification of lymphoproliferative disorders. Many of the lymphocyte specific monoclonal antibodies used, however, can only be applied to frozen tissue sections. In this paper we report the successful application of a number of these antibodies to paraffin processed tissue utilizing alternative fixatives and the highly sensitive immunogold-silver staining method. The best fixatives for this purpose were formol dichromate, periodate-lysine-paraformaldehyde (PLP) and a novel fixative formed from the addition of a dichromate solution to PLP.     

Immunohistological detection of human cytotoxic/suppressor T cells using antibodies to a CD8 peptide sequence.

AIMS: To evaluate whether cytotoxic/suppressor T cells can be detected in paraffin wax embedded human tissue samples using antibodies to a synthetic CD8 peptide sequence. METHODS: Polyclonal and monoclonal antibodies were raised against a 13 amino acid peptide sequence from the cytoplasmic portion of the alpha chain of the human CD8 molecule. RESULTS: These antibodies specifically detected the native form of the CD8 polypeptide when tested by immunoprecipitation with radiolabelled T cells, and gave the expected staining pattern for cytotoxic/suppressor T cells in cryostat sections. Being raised in rabbits, the polyclonal antibodies were also useful for double labelling for CD8 in conjunction with monoclonal antibodies. CD8 positive cells could also be detected in paraffin wax embedded tissues. This was achieved without prior treatment of the sections if the tissue had been fixed in Bouin's fixative. When tissues had been exposed to conventional formalin fixation, preliminary microwave treatment was required. CONCLUSIONS: These findings provide further evidence that antibodies against leucocyte associated antigens, capable of reacting on paraffin wax embedded tissue, can be produced by immunisation with synthetic peptide sequences.     

Enzyme-labeled Antigen Method: Factors Influencing the Deterioration of Antigen-binding Activity of Specific Antibodies during Formalin Fixation and Paraffin Embedding

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.     

Use of freeze-dried paraffin-embedded sections for immunohistologic staining with monoclonal antibodies

Most diagnostically valuable monoclonal antibodies recognize antigens that do not survive conventional tissue processing. The use of frozen tissue sections for immunohistologic studies overcomes this obstacle but introduces a number of practical problems, e.g., the necessity to store material in the frozen state, poor morphologic preservation, etc. In the present paper we report that antigenic denaturation during conventional tissue processing appears to occur during exposure to aldehyde-containing fixatives and to alcohol but not as a result of heating or exposure to melted paraffin wax. In consequence, we have developed a technique by which tissue is freeze-dried and then embedded directly in paraffin wax. All but one of the 40 monoclonal antibodies investigated stained the freeze-dried paraffin sections with an intensity equal to or greater than that observed on frozen sections. There was less diffusion artifact and less background staining than in cryostat sections, and cellular morphology was better preserved. One of the most important advantages of this new method is that antigens in freeze-dried paraffin-embedded tissue are stable, and tissue blocks may be handled in the same manner as conventional paraffin blocks. An additional finding was that, once the tissue has been freeze-dried, paraffin embedded, and sectioned, the antigens it contains are resistant to fixatives (e.g., formol, formol sublimate, alcohols) which would very rapidly cause their destruction in frozen sections.     

The Effect of Various Fixatives and Trypsin Digestion Upon the Staining of Routine Paraffin-Embedded Sections by the Peroxidase-Antiperoxidase and Immunofluorescent Technique

Abstract

The use of immunohistochemical techniques in biological and diagnostic pathology laboratories, and the theory of the unlabelled peroxidase-antiperioxidase technique, is briefly reviewed. The results of experiments using a variety of routine fixatives, with pretreatment of the sections with a trypsin solution, indicated that while Bouin's fixative alone was superior to formalin fixation, the pretreatment of sections with a trypsin solution gave markedly improved results with either fixative — the results with buffered formalin then being almost equal to those after Bouin's fixative. This methodology allows both prospective and retrospective immunohistochemical studies to be made on routinely fixed and processed paraplast-embedded tissues using the light microscope.     

Semiquantitative analysis of the effects of fixation and paraffin embedding on immunoreactivity of renal basement membranes to a monoclonal antibody against type IV collagen.

The effects of formalin fixation and paraffin embedding on the immunoreactivity of human kidney to a monoclonal anti-type IV collagen antibody (JK-199) were examined semiquantitatively by a modified enzyme-linked immunosorbent assay (ELISA). The intensity of immunoreactivity in paraffin sections of the tissue fixed overnight with 10% formalin was approximately 70% of that in frozen sections. Immunoreactivity reduced to this extent did not impair the specific staining of basement membranes. Paraffin sections of tissues fixed 2 days showed 50-60% of the reactivity in the frozen sections of the tissue fixed overnight; the basement membranes in Bowman's capsules were stained positively, but those in other sites were not. The paraffin sections of tissues fixed 7 or 14 days showed no specific immunostaining. The immunoreactivity for type IV collagen in the basement membranes was restored after treatment with pronase E. The immunoreactivity after the enzymatic treatment was about 150% of that in the frozen sections of the overnight fixed specimens.     

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.     

Human intestinal mucosal mast cells: evaluation of fixation and staining techniques

The staining properties of tissue mast cells are influenced by the method of fixation. Differences in fixation and staining techniques may explain the contradictory results in the published reports on the number of human mucosal mast cells (MMC) in the gastrointestinal mucosa in health and disease. We have examined the influence of fixatives on the staining properties of human MMC in operative biopsy specimens of human jejunum. Specimens were divided into pieces, each of which was fixed in one of the following fixatives: Carnoy's, basic lead acetate (BLA), Baker's, Bouin's, isotonic formol-acetic-acid (IFAA), 10% neutral buffered formalin, formol sublimate, and formol saline. Thereafter, tissues were paraffin-embedded and 5 micron sections were cut and stained with either astra-blue/safranin pH 0.3, or toluidine blue pH 0.5. Counts of the number of MMC/mm2 were obtained for each fixation method. The results show a critical influence of the fixative on the number of mast cells identified after staining. For example with astra-blue/safranin the mean MMC/mm2 count was 40 in formol-saline-fixed specimens, and 268 in Carnoy's-fixed specimens. In biopsies fixed with formalin-based fixatives, mast cells were more readily stained with toluidine blue. It is recommended that Carnoy's or BLA be used as the fixative for any light microscopic study of human MMC.     

Using Kryofix as alternative for formalin results in more optimal and standardized immunostaining of paraffin sections.

Although used for over one century formalin has several disadvantages which Kryofix, an alternative fixative for paraffin blocks used in Leiden for 6 years, does not have. In this study the effects of Kryofix on tissue regarding immunohistochemistry are compared with those of buffered formalin. All markers studied showed enhanced staining in the Kryofix blocks after 4 hours of fixation, whilst in some cases the immunostaining of the formalin blocks was even negative. For all markers, the sera could be further diluted for the Kryofix sections, for some with as much as a factor 20, enhancing the cost-effectiveness of the method. We established that for formalin, the fixation time strongly influenced the results. For Kryofix there was no time factor: the immunostaining results of 1 hour fixation were identical to those after 3 months of fixation. This study shows that by this method of fixation, in which there is no cross-linking of proteins, immunostaining of paraffin sections is optimized and standardized.     

Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies.

AIMS--To evaluate the effect of microwave irradiation on the staining quality of a range of commonly used primary antibodies in archival, formalin fixed, paraffin wax embedded material, with emphasis on antibodies that have previously worked successfully only on frozen tissue. METHODS--Immunocytochemistry (streptavidin-biotin complex technique) was performed on histological sections of a range of normal and pathological tissues, after varying treatment with microwave irradiation. The staining quality of each antibody was compared with that achieved without prior treatment of the sections or after enzyme predigestion. RESULTS--Microwave irradiation permitted successful immunostaining with 20 antibodies that stained only frozen tissues before. The staining characteristics of 21 antibodies that were already known to stain formalin fixed, paraffin wax embedded material were improved. Another 39 antibodies did not show enhanced staining with microwave irradiation. The method preserves tissue morphology and produces more consistent staining than that achieved by enzyme predigestion with many antibodies. Microwave irradiation may also allow some primary antibodies to be used at higher working dilutions. The citrate buffer used in this study avoids the necessity of exposure to heavy metal salts. CONCLUSIONS--Microwave antigen retrieval represents an important technical advance within immunocytochemistry that will greatly increase the range of antibodies which can be used to study formalin fixed, paraffin wax embedded tissues.     

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.     

Demonstration of immunoglobulin in cryostat and paraffin sections of human tonsil by immunofluorescence and immunoperoxidase techniques. Effects of processing on immunohistochemical performance of tissues and on the use of proteolytic enzymes to unmask antigens in sections.

A fluorescein isothiocyanate (FITC) technique and one based on peroxidase-antiperoxidase (PAP) were used to study the distribution of immunoglobulin (Ig) in cryostat and paraffin sections of human tonsil. Trypsin and other proteolytic enzymes were used to 'unmask' the antigen in paraffin sections. The effects of processing, and particularly of fixation, on the immunohistochemical response of tissues were studied. The FITC and PAP methods detected Ig in paraffin and cryostat sections equally well. The distribution of the antigen was the same with both methods but the PAP method was the more informative. Formaldehyde-sucrose solution proved more suitable for fixing tissues for immunohistochemistry than glutaraldehyde. Trypsin revealed antigen in parraffin sections more efficiently than pepsin, papain, or pronase. Surface Ig (s-Ig) could be demonstrated in trypsinised paraffin sections but less effectively than in cryostat sections. Trypsinised paraffin sections were, however, more suitable for intracellular Ig (c-Ig) than cryostat sections although the performance of cryostat sections could be improved by prior fixation with a coagulative fixative.     

Microwave fixation: in situ tick (Acari: Ixodidae) histoanatomy, thin sectioning of tick tissues, and antigen preservation in mouse spleen

Microwave irradiation was used for the fixation of eggs, nymphs, and adult Boophilus spp. ticks. Although optimal temperatures for fixation of the different tick stages varied, heating to 58 degrees C of adult ticks submerged in either PBS or fixative was found to be sufficient. After microwave fixation, whole adult ticks, hand held, were sectioned with a sharp razor blade. The resulting sections revealed the in situ histoanatomy of the tick. Thin sections of ticks were obtained after either paraffin or polyester wax embedding. Microwave fixation combined with polyester wax embedding made serial thin sections of the different stages of Boophilus ticks possible. The technique preserved antigens as demonstrated by the immunostaining of lymphocytes and erythrocytes infected with Babesia microti in mouse tissues subjected to the same treatment as the ticks. With the microwave fixation-polyester wax technique, the specimen preparation time from fixation to the section on the glass slide was reduced to less than 8 h.     

An evaluation of potentially suitable fixatives for immunoperoxidase staining of estrogen receptors in imprints and frozen sections of breast carcinoma

The estrogen receptor (ER) content of breast carcinoma is generally accepted as valuable in predicting clinical outcome and tumour response to hormonal manipulation. We applied a new immunocytochemical assay for estrogen receptors (Abbott ERICA Monoclonal) to 20 breast tumours, and examined the efficacy of 16 fixation procedures before immunoperoxidase staining of frozen sections and imprint preparations. Our findings indicate that the fixatives of choice are periodatelysine-paraformaldehyde at 22 degrees C, or 10% formalin followed by acetone at -10 degrees C. These fixation procedures are simpler, less time-consuming, and provide superior staining, tumour cytomorphology and higher ER values than the 3-reagent sequence recommended by Abbott Laboratories. There was a significant correlation between the ER scores in the frozen sections and the imprints. Positive ER cytosol results correlated with the staining index in the frozen sections, and the ER scores in the imprints. We conclude that imprints are suitable preparations for ER analysis by the immunoperoxidase technique, particularly for small tumour specimens.