Observations on the results of specific histochemical techniques and empirical staining methods on several tissue/organ types using a variety of fixing fluids

Several histotechniques (Harris hematoxylin and alcoholic eosin, Gomori's reticulum method, Verhoeff's elastic stain; Gomori's trichrome, Mallory's aniline blue collagen stain and the Cameron and Steele method) were each used with a number of fixatives (Lavdowsky's chromic acid, Karnovsky's glutaraldehyde/paraformaldehyde, neutral buffered formalin, Lavdowsky's formalinacetic acid-alcohol, Zenker's and Helley's). Lavdowsky's chromic acid fixative provided superior results as compared to the other fixing fluids and its use was critical with respect to the trichrome or connective tissue staining procedures. The Cameron and Steele modification used with Lavdowsky's chromic acid fixative gave the best differentiation of tissue types and was also the most reproducible.     

Silver Acetate Autometallography: An Alternative Enhancement Technique for Immunogold-Silver Staining (IGSS) and Silver Amplification of Gold,Silver, Mercury and Zinc in Tissues

Abstract

Silver lactate autometallography in immunogold silver staining (IGSS) usually requires development in darkness to avoid excessive background staining. Our alternative method of silver enhancement of IGSS on paraffin sections from Bouin's or formalin fixed pancreas uses silver acetate in combination with hydroquinone in low pH buffer. The modification was tested with a range of antibodies in normal and diseased tissues (all routinely fixed and paraffin embedded), in acetone postfixed cryostat sections, and in semithin sections of glutaraldehyde fixed and resin embedded tissues. Silver acetate autometallography was also tested in various systems for the visualization of tissue metals like sulfides and selenides of mercury and zinc, silver, and gold. Comparisons between sections exposed to silver lactate and the silver acetate developer showed no significant difference in the number of structures stained. The degree of background staining was often lower when silver acetate was to used as the ion donor, especially with IGSS. The advantage the technique described here is that the development process can be controlled, using normal bright field light microscopy. (The J Histotechnol 11:213, 1988.)     

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.     

Differential staining of mast cells with toluidine blue

Abstract

Mast cells play a significant role in inflammatory diseases such as asthma, inflammatory bowel disease, and autoimmune diseases. Inhibition of c-kit receptor tyrosine kinase, a growth factor receptor, significantly reduces mast cell numbers. The purpose of this study was to determine the effect of Compound X (a c-kit inhibitor) on mast cell numbers in rats. Connective tissue mast cells (CTMCs) and mucosal mast cells (MMCs) have differing histochemical characteristics which presents a challenge when staining for quantification by semi-automated image analysis. CTMCs are present in tissues such as tongue and skin and will stain readily in tissues fixed routinely. In contrast, MMCs, such as those present in the intestinal mucosa, are sensitive to fixation. Brief fixation in Carnoy’s solution, although seldom used due to its composition (a mixture of ethanol, chloroform, and acetic acid), was employed to fix tissues for MMC staining, while tissues for CTMC demonstration were fixed in 10% neutral buffered formalin. An enzyme histochemistry method, napthol AS-D choloroacetate (specific esterase), was briefly considered for staining; however, granulocytes stained along with mast cells, requiring manual identification and exclusion, thereby rendering the method incompatible with automated means of quantification. Instead, staining was performed using two different toluidine blue methods which have proven conducive to semi-automated image analysis techniques. CTMCs were stained using Luna’s toluidine blue, while MMCs were stained with Matsson’s toluidine blue modification. In summary, the selected methods, based upon a conventional stain, were easy to do and successfully identified both populations of mast cells for quantification by image analysis.     

3D approach visualizing cellular networks in human lymph nodes

Lymph node diagnostics are essentially based on cutting thin sections of formalin fixed tissues. After hematoxylin and eosin stain, Giemsa stain and immunohistochemical staining of these tissues, the lymph node diagnosis is done using a light microscope, looking at two-dimensional pictures. Three-dimensional visualizations of lymph node tissue have not been used in lymphoma diagnostics yet. This article describes three-dimensional visualization of lymphoid tissue, using thick paraffin sections, immunostained with monoclonal antibodies, confocal laser scanning and data processing with appropriate software and the 3D printing process itself. The advantages and disadvantages of different printing techniques are discussed as well as the application of 3D models in diagnostics, teaching and research of lymph nodes.     

Mast cell heterogeneity in dog skin

The degree of metachromasia of mast cell granules is known to vary with the type of tissue fixation and among different tissues and species. The present study sought to determine whether mast cells in dog skin are heterogeneous with respect to fixation and staining properties. We performed skin biopsies in six anesthetized, atopic dogs and one mongrel dog. One biopsy was fixed in formalin and a second, from a parallel abdominal site, was fixed in basic lead acetate (Mota's solution). Adjacent sections from each biopsy were stained with alcian blue (1%, pH 0.5) or for chloroacetate esterase activity. In alcian blue-stained sections, one-third fewer mast cells were detected in skin fixed in formalin (1,836 +/- 454 mast cells/mm3, mean +/- SEM) than in skin fixed in basic lead acetate (2,684 +/- 527 mast cells/mm3) (P less than 0.05). The chloroacetate esterase reaction detected the larger number of mast cells regardless of the fixative used. We conclude that mast cell heterogeneity, as demonstrated by metachromatic staining following different types of tissue fixation, exists in dog skin. "Typical" mast cells stain with alcian blue regardless of fixation; however, "atypical" mast cells exhibit metachromasia only after fixation in basic lead acetate. Both the typical and atypical types of mast cells have chloroacetate esterase activity.     

Staining efficacy assessment of a differential routine and special stains for pathological stromal calcifications in maxillofacial lesions

ABSTRACT

Head and neck connective tissue lesions may have diverse calcifications within the fibrous connective tissue stroma. The perplexity involved in the identification and determination of the nature or degree of calcification through routine hematoxylin and eosin (H&E) stains necessitates the usage of a specific, simple, and cost- and time-effective differential staining techniques. The aim of the present study was to develop criteria to distinguish bone formation from bone resorption using methylene blue-acid fuchsin (MB/AF) stain and the role of collagen fibers in the identification of stromal calcifications using polarizing microscopy with picrosirius red stain. Twenty cases with pathological diagnoses for various stromal calcifications in maxillofacial lesions were retrieved from the departmental archives. Decalcified formalin fixed paraffin embedded tissue sections were stained with hematoxylin and eosin, Masson’s trichrome (MT), methylene blue-acid fuchsin (MB/AF), and picrosirius red. The stained sections were assessed to identify the calcifications found in the surrounding connective tissue stroma. It was observed that most cases showed maximum staining intensity with MB/AF stain as compared to the other staining methods. Moreover, the results suggested that contrast between calcification and stromal soft tissue was best distinguished with the MB/AF stain except in the case of dystrophic calcifications. Along with this, polarizing microscopy with picrosirius red enables better characterization of stromal components. Although the H&E stain and a connective tissue stain i.e. Masson’s trichrome, are employed routinely in histopathology; the use of special stains such as MB-AF and picrosirius red facilitates the identification of calcifications from the stromal tissues.     

Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.

Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions.     

Evaluation of histochemical staining techniques for visualization of bacterial biofilm-associated extracellular matrix in skin

Biofilms play a significant role in histopathology and are complex structures consisting of bacterial cells embedded in an extracellular matrix that contains polysaccharides, proteins, and deoxyribonucleic acid (DNA). The biofilm matrix limits the effectiveness of topical antibiotic treatment in infected wounds and impedes wound healing and immune responses. The purpose of this study was to visualize the biofilm-associated extracellular matrix using standard histological techniques. The commercially available MatTek epidermal full-thickness skin tissue model (EFT-400) was injured and infected for 24 h with biofilm-forming Staphylococcus aureus. Tissue for paraffin sections was fixed in formalin, microwave-processed, and embedded in paraffin. Serial sections cut to 5 microns were stained with Periodic acid-Schiff reagent, Calcofluor, modified Congo Red/Ziehl carbol fuchsin stain and Feulgen reaction. Stained tissues were evaluated using light and fluorescent microscopy. A detailed analysis of the application of the different staining techniques in demonstration of biofilm-associated extracellular matrix revealed that both carbohydrates and DNA were present. Discussion of the value of each staining technique is presented.     

Immunohistochemical assays in prostatic biopsies processed in Bouin's fixative

AIMS: To investigate the problems involved in undertaking immunohistochemistry (IHC) and nuclear morphometry using Bouin's fixed prostate biopsies. METHODS: Archival Bouin's fixed and formalin fixed, paraffin wax embedded prostatic biopsies were immunostained for three nuclear biomarkers (minichromosome maintenance protein 2 (MCM-2), p27, and Ki-67), one membrane localised biomarker (C-erb-B2), CD34, and alpha methylacyl-CoA racemase (AMACR). The quality of IHC staining was compared between tissues prepared separately in both fixatives. Feulgen staining was also performed on Bouin's fixed tissues to check its suitability for nuclear morphometry. RESULTS: MCM-2 staining was completely negative in Bouin's fixed tissues, whereas p27 showed more background and excess cytoplasmic staining in Bouin's fixed versus formalin fixed tissues. C-erb-B2 showed non-specific, strong luminal cell staining in the Bouin's fixed tissue. Feulgen staining was also very weak in Bouin's fixed tissue. However, Ki-67, AMACR, and CD34 worked equally well in Bouin's and formalin fixed tissues. CONCLUSIONS: Bouin's fixed tissues may be unsuitable when subsequent IHC and morphometry are contemplated. An awareness of which antibodies are suitable for use in Bouin's fixed biopsies is essential.     

Immunohistochemical staining of colorectal tissues with monoclonal antibodies to ras oncogene p21 product and carbohydrate determinant antigen 19-9.

Two monoclonal antibodies were applied to benign, dysplastic, and malignant human colorectal tissues using immunohistochemical techniques on formalin fixed paraffin embedded material. RAP-5 antibody is directed against a synthetic peptide, reflecting an amino acid sequence of the ras oncogene p21 protein product. Despite using several different techniques and antibody dilutions differential staining between the various epithelial populations was not obtained. RAP-5 also showed other tissue components such as plasma cells, histiocytes, fibroblasts, smooth muscle and vascular endothelium. CA19-9 antibody recognizes an epithelial surface carbohydrate antigen originally derived from a human colorectal carcinoma cell line: it did not stain normal colorectal mucosa or adenomatous polyps, but showed focal expression of variable strength in regenerative, dysplastic, and cancerous mucosa in ulcerative colitis, and in non-colitic colorectal carcinoma. Neither antibody was found to be a reliable marker of the evolution of malignant mucosal changes, although CA19-9 may be of limited use in confirming adenocarcinoma of gastrointestinal origin.     

Comparison of formalin, buffered formalin, and Bouin's fixation on the detection of human papillomavirus deoxyribonucleic acid from genital lesions

Detection of nucleic acid sequences homologous to human papillomavirus (HPV) relies primarily on their extraction from unfixed tissue. We detected HPV sequences in DNA extracted from paraffin-embedded tissue fixed in formalin (buffered and unbuffered) and Bouin's solution by dot blot hybridization. A detectable hybridization signal was noted in 32% of these fixed tissues which were chosen from cases where HPV DNA was detected in the unfixed tissue. When using a homologous 32P-labeled probe and a high stringency wash, the hybridization signal was lost if DNA was extracted after Bouin's fixation and diminished after formalin fixation, more so with unbuffered formalin. Similar differences in the hybridization signals among the different fixatives after high stringency wash were noted with in situ hybridization. Southern blot analysis showed that DNA extracted from tissues fixed in Bouin's was degraded and ranged in size from 100 to 500 base pairs as compared with 100 to 900 base pairs for DNA extracted from tissue fixed with unbuffered formalin. In contrast, no degradation was noted after fixation with buffered formalin. These results demonstrate that HPV sequences can be identified in DNA extracted from paraffin-embedded, fixed tissue. However, use of some fixatives may preclude identification of HPV type, by either dot blot or in situ hybridization.     

A combination Prussian blue – hematoxylin and eosin staining technique for identification of iron and other histological features

The Prussian blue reaction (PB) detects ferric iron in histological sections but the nuclear fast red (NFR) counterstain does not selectively stain the surrounding tissue and cellular features very well. The PB/NFR stain has the advantage of detecting iron located in tissue sections, but a significant disadvantage of having poorly differentiated tissue components, as compared to a routine hematoxylin and eosin (H&E). We developed a combination of Gomori’s Prussian blue/H&E staining method (PB/H&E), and modified the technique for best performance and clarity, then assessed the ability of this new combination stain to differentiate histological features of the tissue and identify iron. Serial sections from seven formalin fixed paraffin-embedded liver samples previously diagnosed with the presence of ferric iron were subjected to our routine H&E, routine PB/NFR and three trials of the new Prussian blue/H&E combination (PB/H&E). The technique that best differentiated the histological components of tissues containing iron was further tested on liver sections from a variety of species to verify consistency i.e. equivalence in staining intensity, concentration, brightness between sections of the same sample and quality i.e. coloration, vividness, recognizable differentiation of tissue components, improved staining.     

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.     

Some Effects of pH,Aldehyde Fixatives and Exposure Times on Tissues Prepared for Light Microscopy

Abstract

A study was conducted to determine some effects of pH, exposure times and aldehyde components of fixatives on tissue preservation. To ascertain simultaneous effects on a variety of tissues, whole snails were fixed in buffered and nonbuffered solutions of 2% glutaraldehyde or 2% formaldehyde (made from commercial formalin). Tissues were exposed for two, 12 and 24 hours, subsequently dehydrated, and embedded in polyester wax. Sections were cut 6 μm in thickness, and samples from each of the treatments were simultaneously stained with Lehman's polychrome stain. Results suggest that 2% nonbuffered formaldehyde is unsatisfactory, at all exposure times, but buffering improves the quality of preservation. Glutaraldehyde fixation, both buffered and nonbuffered, gave good to excellent preservation at all exposure times. Buffered glutaraldehyde at moderate (2–12 hour) exposure times resulted in excellent preservation and stainability of a variety of tissues. However, lengthy exposure times resulted in alteration of molecular components, as judged by altered staining qualities of tissues and cellular components     

Three-dimensional architecture of the intrinsic tongue muscles using a modified alkaline maceration method

The three-dimensional architecture of the intrinsic tongue muscle fibers using the anterior part of the rabbit tongue was studied by scanning electron microscopy with a modified chemical-maceration method. The tongue tissues fixed with 10% formalin solution were treated with 1% OsO4 solution at 5 minutes for hardening of the specimen surface. Subsequently, they were immersed in 6N-NaOH solution for 30 minutes at 60 degrees C for the removal of connective tissues followed by dissection of muscle fibers under a binocular microscope to clarify the structure of the intrinsic tongue muscles. The specimens were treated with tannic acid and OsO4 (conductive staining method; Murakami, 1974), and observed with a SEM. Muscular fiber bundles of the transverse and vertical muscles of the tongue changed their direction at the alignment on the sites where the bundles enter the longitudinal muscles from the innermost surface to form monolayers of muscular bundles extending anteroposteriorly. These muscular bundles formed tunnel-like structures each of which covered a longitudinal muscle bundle. It was considered that these tunnel-like structures support the contraction of the longitudinal muscles as the "muscular sheath".     

A staining method of PIVKA-II by an indirect immunoenzyme technic

The localization of PIVKA-II in liver tissue was studied by the immuno-staining method using monoclonal antibody. The cell lines derived from hepatoblastoma (huH-6), hepatocellular carcinoma (PLC/PFR/5), and normal liver (Chang) were used as materials for staining. We succeeded to stain PIVKA-II, when the materials were fixed in a formalin solution and frozen. In patients with hepatocellular carcinoma, PIVKA-II was stained in the cytoplasm of both hepatoma and non-hepatoma cells.     

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.     

Immunohistochemical detection of infectious bursal disease and infectious bronchitis viral antigens in fixed, paraffin-embedded chicken tissues

Immunoperoxidase and immunofluorescent techniques were used to detect and localise infectious bursal disease virus (IBDV) and infectious bronchitis virus (IBV) in fixed, paraffin-embedded chicken tissues. A modified Bouin's solution (9 parts of 1% picric acid to 1 part of 10% neutral buffered formalin and 5% acetic acid) proved to be the fixative of choice. Tissues fixed in 10% neutral buffered formalin showed heavy background staining, whereas enzyme treatment of these tissues succeeded in exposing viral antigens and thus giving a specific reaction. For routine diagnostic purposes it will prove advantageous to detect IBDV and IBV in fixed, paraffin-embedded material.     

In situ demonstration of tissue proliferative activity using anti-bromo-deoxyuridine monoclonal antibody.

Immunohistochemical staining with anti-bromo-deoxyuridine (BrdU) monoclonal antibody was performed on a variety of human tissues following in vitro incubation with BrdU. The effect of different fixatives and DNA denaturation techniques on the reactivity with anti-BrdU was investigated. Optimal preservation of the antigenicity of BrdU incorporated into the DNA of proliferating cells was seen in tissues fixed in Bouin's fluid, while samples which had been fixed with cross-linking reagents, such as formalin, were usually unreactive. Positivity for BrdU was restored in formalin fixed tissues after digestion with pepsin, but this was usually associated with loss of morphological details. Acid and thermal DNA denaturation techniques gave similar results. It is concluded that Bouin fixation followed by acid or thermal denaturation of DNA is the method of choice for the in situ detection of cells in S-phase using anti-BrdU monoclonal antibody.