Demonstration of laminin, a basement membrane glycoprotein, in routinely processed formalin-fixed human tissues

Laminin was demonstrated by immunoperoxidase and immunofluorescence staining in sections of normal human tissues fixed in formalin and routinely processed in paraffin. Exposure of the sections to a solution of pepsin (Burns et al. (1980) Histochemistry 67:73-78) revealed the antigenicity of this basement membrane glycoprotein. Sections from paraffin blocks stored for years at room temperature could be stained with this procedure. Normal human tissues, developing fetal tissues and tumors could be stained with this method. The staining patterns were similar to those seen in unfixed frozen sections. It thus appears that basement membrane components can be detected by immunohistological means from routinely processed histological samples, once the sections are pretreated with proteases. Staining for laminin could be used in embryonic studies and in histopathology to study the relation of cells to basement membranes and for the visualization of normal and abnormal vascularization.     

Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application

This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryostat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution. Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal anti-type IV collage antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections. It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Human specific anti-type IV collagen monoclonal antibodies,characterization and immunohistochemical application

Summary This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryosat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution.Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal antitype IV collagen antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections.It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Polychromatic staining of epoxy semithin sections: a new and simple method

A simple, rapid method is described for the polychromatic coloration of semithin sections, which is applicable to material routinely processed for transmission electron microscopy. Material fixed with a glutaraldehyde-paraformaldehyde mixture and postfixed in osmium tetroxide with or without potassium ferrocyanide and embedded in different types of resin (Durkupan-ACM, Spurr resin, Taab resin) can be used. Constant and homogenous results are obtained with this technique, the staining procedure being achieved at room temperature in no more than 10 min. Sections of 0.5–1 m in thickness are oxidised and bleached. After washing, sections are stained in two steps with carbol methylene blue/carbol gentian violet solution and pararosaniline solution. Using the method described in this paper, a polychromatic coloration of the different cells and tissues was obtained (epithelial cells in various shades of blue-violet, connective tissue and elastic laminae of blood vessels in pink or red, etc.). This procedure provides greater contrast between cytoplasm and nuclei, and among the different types of cells and tissues than is seen with toluidine blue, which is very useful for observation and photography of semithin sections. Polychromatic methods found in the literature are normally complex and require a lengthy staining time or cannot be applied on material routinely processed for transmission electron microscopy. Our method is simple, rapid and can be used on any type of material routinely processed for transmission electron microscopy and embedded in epoxy resins.     

Comparison of monoclonal antibodies PC 10 and MIB 1 on microwave-processed paraffin sections

Abstract. Antibodies against the proliferation-associated nuclear antigen (PCNA) and against the Ki-67 protein are widely used as operational proliferation markers in human tumour diagnostics. The original Ki-67 antibody had the inherent drawback in that it could only be used when fresh-frozen material was available. The antibody PClO was supposed to offer the advantage that it could be applied on formalin-fixed and paraffin-embedded tissues. However, in cases in which the formalin fixation exceeded 4 h, PC10 staining proved to be inconsistent and often failed.
The aim of this study was to compare a recently prepared Ki-67 equivalent monoclonal antibody (MIB 1) and PC10 in routinely fixed histopathological material using antigen retrieval by microwave processing.
Antibody MIB 1 stained the nuclei of cells known to belong to the proliferative compartments in microwave-processing paraffin sections of formalin-fixed tissues. Quiescent cells were consistently negative for MIB 1 staining. In contrast, PC10 was positive in almost all nuclei of different tissues in microwave-treated paraffin sections. Thus, antigen retrieval by microwave processing is beneficial for the detection of the Ki-67 protein in paraffin sections, whereas it is not needed for the detection of the PCNA.     

Immunofluorescent Staining of Leptospires in Pepsin Treated Histologic Sections

A standard immunofluorescent method was modified for the staining of leptospires in formalin fixed, paraffin embedded tissues. Routine histologic sections were deparaffinized and treated with pepsin prior to staining. Pepsin treatment greatly enhanced subsequent staining of leptospires in naturally infected bovine and porcine tissues as well as in artificially infected tissues. Leptospires in naturally infected bovine tissues were usually undetectable in untreated sections but clearly visible in stained pepsin-treated sections. Naturally infected porcine kidney usually contained high levels of leptospiral antigen which could be stained without prior pepsin treatment. However, pepsin treatment of porcine tissues greatly increased the amount of leptospiral antigen detectable and made individual leptospires more conspicuous. The staining method could employ a single antiserum for the staining of leptospires from 13 serogroups. Also, leptospires could be stained in tissues stored in formalin for more than 14 months and in 26-year-old paraffin embedded tissues.     

Immunofluorescent staining of leptospires in pepsin treated histologic sections

A standard immunofluorescent method was modified for the staining of leptospires in formalin fixed, paraffin embedded tissues. Routine histologic sections were deparaffinized and treated with pepsin prior to staining. Pepsin treatment greatly enhanced subsequent staining of leptospires in naturally infected bovine and porcine tissues as well as in artificially infected tissues. Leptospires in naturally infected bovine tissues were usually undetectable in untreated sections but clearly visible in stained pepsin-treated sections. Naturally infected porcine kidney usually contained high levels of leptospiral antigen which could be stained without prior pepsin treatment. However, pepsin treatment of porcine tissues greatly increased the amount of leptospiral antigen detectable and made individual leptospires more conspicuous. The staining method could employ a single antiserum for the staining of leptospires from 13 serogroups. Also, leptospires could be stained in tissues stored in formalin for more than 14 months and in 26-year-old paraffin embedded tissues.     

Cryofixed,freeze-dried and paraffin-embedded skin enables successful immunohistochemical staining of skin basement membrane antigens

Summary Conventional chemical fixation and paraffin-embedding procedures give good preservation of morphology, although the antigenicity of many proteins in the tissue sample is destroyed. On the other hand, fresh frozen sections can preserve the antigenicity, but provide poor morphological preservation. To overcome this dilemma, cryofixation and freeze drying were used on human skin tissue, applying methodology which has only been used to study lymphoid tissue. First, fresh human skin was cryofixed in liquid isopentane (–160° C) cooled by liquid nitrogen. The skin was then freeze-dried at –40° C and 10^–2 atmospheric pressure for 72 h, followed by embedding in paraffin. Sections 4 m thick taken from this cryofixed, freeze-dried, and paraffin-embedded skin were stained with hematoxylin-eosin or used for immunolabeling with antibodies against basement membrane antigen, including type IV and type VII collagen, bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and GB3 antigen. The morphological preservation of these sections was as good as that of routine formalin-fixed and paraffin-embedded skin sections. The basement membrane was clearly immunostained with all antibodies used, and the intensity of the reaction was as strong as that seen in frozen sections. Evaluation of antigen distribution in conjunction with the detailed skin structure was therefore possible in the same sections.A part of this work was presented at the 90th Annual Meeting of the Japanese Dermatological Association, Kyoto, Japan, April, 1991     

Detection of peroxisomes in human liver and kidney fixed with formalin and embedded in paraffin: the use of catalase and lipid beta-oxidation enzymes as immunocytochemical markers

Summary We describe the immunocytochemical localization of four peroxisomal enzymes by light microscopy in human liver and kidney processed routinely by formalin fixation and paraffin embedding. Monospecific antisera against catalase and three enzymes of peroxisomal lipid -oxidation (acyl-CoA oxidase, bifunctional protein (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase) and 3-ketoacyl-CoA thiolase) were used in conjunction with either the indirect immunoperoxidase method or the protein A—gold technique followed by silver intensification. The sections of formalin-fixed paraffin-embedded tissue had to be deparaffinized and subjected to controlled proteolysis in order to obtain satisfactory immunostaining. Under the conditions employed, peroxisomes were distinctly visualized in liver parenchymal cells with no reaction in bile duct epithelial or sinusoidal lining cells. In the kidney, peroxisomes were confined to the proximal tubular epithelial cells with negative staining of glomeruli, distal tubules and collecting ducts. A positive immunocytochemical reaction was obtained even in paraffin blocks stored for several years. The method offers a simple approach for detection of peroxisomes and evaluation of their various enzyme proteins in material processed routinely in histopathology laboratories and should prove useful in the investigation of the role of peroxisomes in human pathology for both prospective and retrospective studies.     

Evaluation of Preparation, Staining and Microscopic Techniques for Counting Incremental Lines in Cementum of Human Teeth

Apposition of cementum occurs in phases resulting in two types of layers with different optical and staining properties that can be observed by light microscopy. Narrow, dark staining incremental lines are separated by wider bands of pale staining cementum. The distance from one line to the next represents a yearly increment deposit of cementum in many mammals, and counting these lines has been used routinely to estimate the age of the animals. Incremental lines in cementum have also been observed in sections of human teeth, and the object of the present investigation was to examine a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, either ground or decalcified, were cut from formalin fixed human dental roots, paraffin embedded or frozen, and stained using several techniques. The cementum was investigated using conventional light, fluorescence, polarized light, confocal laser scanning, interference contrast, phase contrast, and scanning electron microscopy. Incremental lines in the cementum could be observed in ground sections and, following decalcification, in both frozen and paraffin embedded sections. Toluidine blue, cresyl violet, hematoxylin, or periodic acid Schiff (PAS) stained incremental lines allowing differentiation by conventional light microscopy. Contrast was best using fluorescence microscopy and excitation by green light since the stained cemental bands, but not the incremental lines, fluoresced after staining with cresyl violet, PAS or hematoxylin and eosin. The results with other microscopic techniques were unsatisfactory. Since incremental lines are not destroyed by acids and stain differently than the remaining cementum, it is likely that they possess an organic structure which differs from the cementum. Incremental lines in human dental cementum could be observed best using decalcified sections stained with cresyl violet excited by green light.     

Immunohistochemical localization of sodium-potassium ATPase in human normal stomach and gastric adenocarcinomas

We studied the expression pattern of Na, K-ATPase beta 1 subunit in human normal stomachs and in gastric adenocarcinomas by using anti-Na, K-ATPase beta 1 subunit-specific monoclonal antibody. Tissue samples were processed in formalin solution or in a cold acetic acid-ethanol solution, routinely processed, embedded in paraffin, and an immunoperoxidase method for Na, K-ATPase beta 1 subunit was performed. After antigen retrieval using a steamer in citrate buffer (pH 6.0), tissue sections initially fixed in cold acetic acid-ethanol showed intense immunoreactivity with the antibody at the lateral or basolateral cytoplasmic membrane of normal gastric epithelial cells, at the cytoplasmic membrane of gastric carcinoma cells according to the level of differentiation, and at the cytoplasmic membrane and in the cytoplasm of Schwann cells and neurons in the mesenteric plexus of the gastric wall. Acetic acid-ethanol and paraffin embedding is a useful method for the investigation of the immunohistochemical localization of Na, K-ATPase in normal and diseased tissues.     

Immunostaining of growth hormone and prolactin in paraffin-embedded and stored or previously stained materials.

In attempts to evaluate immunocytochemically autopsy and biopsy material previously obtained and processed for conventional histologic staining, we had to resort to immunostaining of tissues embedded years ago or even sections already stained with hematoxylin-eosin or aldehyde thionin-PAS-orange G. Hypophysial growth hormone and prolactin proved remarkably resistant to such prior treatment with regard to their antigenic properties, and could be readily immunostained in tissue embedded in paraffin 3-4 years earlier, and after destaining of sections prepared up to 7 years earlier. The results of such "retrospective" immunocytochemical evaluation of autopsy and biopsy materail is illustrated with the staining of "pregnancy cells" for prolactin in the hypophysis of a woman postpartum, the immunostaining for prolactin in the cells of adenomas associated with marked hyperprolactinemia, the staining for growth hormone in adenomas removed from children with gigantism, and the immunostaining for prolactin, growth hormone or both in several adenomas that were discovered at autopsy and not associated with a known clinical history of endocrine aberrations.     

TGF-β Receptor Expression in Human Odontoblasts and Pulpal Cells

Transforming growth factor- (TGF-) isoform expression by odontoblasts leads to their sequestration within the dentine matrix, from where they may be released during caries and participate in the reparative processes. Two receptor types for TGF- have been implicated in TGF- induced signalling. The aim of this study was to characterise immunohistochemically the expression of these receptors in sound and carious human teeth to facilitate our understanding of the ability of these cells to respond to TGF- stimulation. Sound and carious human teeth were routinely processed and paraffin sections stained for TGF- receptors I and II, using the StrAviGen immunoperoxidase method. Strong specific staining for both receptor types was observed in the odontoblasts of healthy teeth with the greatest intensity seen with receptor I. Staining of weaker intensity was also observed for both receptors in the underlying cell rich area and pulp core. Similar patterns of staining were observed within carious tissues. We conclude that odontoblasts and other cells of the pulp of mature human molar teeth show the presence of both TGF- receptors I and II in health and disease with odontoblasts showing the strongest expression. Such findings may be important in the response of these cells to tissue injury.     

Investigation of Tissue Preparation Conditions for Non-radioactive In Situ Hybridization: Localization of Transforming Growth Factor-α Message in Rat Kidney

A non-radioactive method of in situ hybridization was used to localize transforming growth factor- mRNA in epithelial cells of collecting ducts and tubules in rat kidney tissue sections. The intensity and specificity of staining were assessed under a variety of tissue preparation conditions, including a direct comparison of paraffin against frozen sections. Under optimal conditions, both the signal strength and the cellular localization of the growth factor message were superior in paraffin sections. The staining method could also be used to localize the message in lung tissue, indicating that the procedure is generally applicable to other tissues. Our results indicate that the use of paraffin sections for non-radioactive in situ hybridization affords a number of advantages for the localization of specific messages in tissue sections.     

Replacing xylene with n-heptane for paraffin embedding

Abstract In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.     

Cytochemical characterization of basement membranes in the enamel organ of the rat incisor

Ameloblasts are unique epithelial cells, in that once they have deposited the entire thickness of enamel and the process of maturation begins, they reform a basal lamina-like structure at their apical surface. In order to characterize further this basal lamina, its composition was analysed using (1) lectin-gold cytochemistry for glycoconjugates, (2) high-iron diamine (HID) staining for sulfated glycoconjugates and (3) immunogold labeling for collagen type IV and laminin. The labeling patterns were compared to that of other more typical basement membranes found in the enamel organ. Sections of rat incisor enamel organs embedded in Lowicryl K4M were stained with Helix pomatia agglutinin (HPA), Ricinus communis I agglutinin (RCA), wheat germ agglutinin (WGA) and Ulex europaeus I agglutinin (UEA). Samples from the late maturation stage were also reacted en bloc with lectins and embedded in Epon for transmission electron microscopic examination or prepared for scanning electron microscopy. Such samples were also stained with HID and conventionally processed for Epon embedding. Tissue sections were then reacted with thiocarbohydrazide-silver proteinate (TCH-SP). Analysis of the lectin labeling suggested that the region of extracellular matrix immediately adjacent to ameloblasts, where the basal lamina is situated, was intensely reactive with HPA and RCA, moderately reactive with WGA, and weakly reactive with UEA. In general, other basement membranes were mildly reactive with all lectins used. No HID-TCH-SP staining was observed directly over the basal lamina while numerous stain deposits were present over other basement membranes of the enamel organ. Immunolocalization of collagen type IV and laminin yielded a weak and variable labeling over the basal lamina. These results are consistent with the concept of basement membrane heterogeneity and, although the precise nature and composition of the basal lamina associated with maturation stage ameloblasts remain to be determined, they suggest that it may possibly function as a specialized basement membrane with particular compositional characteristics.     

Monoclonal antibody analysis of ocular basement membranes during development

As a first step in a study of the role(s) of basement membranes in ocular morphogenesis, we have produced a variety of monoclonal antibodies against native lens capsule from adult chicks, and have used these reagents to stain histological sections of ocular tissues from 4 1/2- to 18-day-old chicken embryos. Four different patterns of immunofluorescence were observed in sections of corneas of 18-day-old chicken embryos stained with these antibodies. The antibodies in group 1 stained the basement membranes of both the corneal epithelium and the endothelium (as well as Descemet's membrane). Those in groups 2 and 3 stained only the epithelial or endothelial basement membranes, respectively. The group 4 antibody stained the corneal stroma as well as Bowman's membrane and Descemet's membrane. The antibodies in group 1 could be further subdivided into groups 1a and 1b on the basis of temporal differences in the onset of staining in corneas from 4 1/2- to 7-day-old embryos. Thus, this series of monoclonal antibodies appears to recognize at least five different antigenic determinants. When these antibodies were used to stain sections of eyes at different stages of development, we found that the characteristic differential staining of some basement membranes was maintained throughout development, while the staining properties of others changed. This indicates that many of the ocular basement membranes may differ from one another in composition or conformation, and that at least some of them may undergo developmental changes. We also noticed a similarity in the pattern of fluorescence associated with the basement membranes of the limbal blood vessels and the corneal endothelium that is consistent with the hypothesis that the corneal endothelium is derived from the early periocular vascular endothelium. Our observations of developing corneas also revealed that the antigen recognized by the group 4 antibody may be produced by both the corneal epithelium and the stromal fibroblasts. The suitability of monoclonal antibodies for probing basement membrane heterogeneity is discussed.     

Replacing xylene with n-heptane for paraffin embedding

Abstract

In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.     

Selective fluorescence of eosinophilic structures in grasshopper and mammalian testis stained with haematoxylin-eosin

After staining with Mayer's haematoxylin and eosin Y, paraffin sections of grasshopper and mouse testis were analysed by both transmitted light and fluorescence microscopy. Under violet-blue (436 nm) light excitation, a bright green emission was observed in all eosinophilic structures. Meiotic spindles (fibres and poles), mitochondrial aggregates, centriolar adjuncts in grasshopper spermatids, the basal lamina, flagellar bundles and remaining cytoplasmic droplets in the lumen of seminiferous tubules showed the most striking fluorescence induced by eosin Y. No emission was found in these structures after haemalum staining. Fluorescent microtubular components also revealed a positive immunoperoxidase reaction for -tubulin. All fixation and embedding procedures (Bouin, Zenker, formaldehyde alone or followed by dichromate or glutaraldehyde, freeze-substitution) were suitable for observation by fluorescence microscopy. Acetylation, deamination, and prolonged washing of stained sections with water, salt solution or ethanol strongly reduced eosin Y fluorescence, while it slightly increased after methylation. These results show that routine haematoxylin-eosin stained tissue sections can be routinely analysed by fluorescence microscopy. The emission of eosin Y allows easy and precise recognition of eosinophilic structures, which are poorly visible under bright field illumination.     

Immunocytochemical demonstration of cytochrome c oxidase with an immunoperoxidase method: a specific stain for mitochondria in formalin-fixed and paraffin-embedded human tissues

Cytochrome c oxidase (CO) is the terminal electron carrier of the respiratory chain and is localized exclusively in the inner membrane of the mitochondria. Using a specific rabbit antiserum against bovine heart CO, mitochondria were specifically stained in sections of formalin-fixed paraffin-embedded human tissues by an immunoperoxidase method. The intensity of the immunostaining in different human tissues correlated mainly with the number of mitochondria and abundance of mitochondrial cristae, i.e., surface area of the inner mitochondrial membrane. Mitochondrion-rich cells, such as myocardial and gastric parietal cells, had very strong cytoplasmic staining. This technique was especially useful for the specific identification of oncocytes in normal tissues and in lesions composed of oncocytes. In addition, the immunoperoxidase method for CO makes possible retrospective investigations of lesions composed of oncocytes, since routine paraffin blocks of formalin-fixed tissue are quite suitable for such studies.