Application of cryo-compatible antibodies to human placenta paraffin sections

The hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) -fixation and paraffin embedding technique has been described to expand possibilities for immuno-labellings due to low denaturation of proteins. In this study, the issue was addressed as to whether the HOPE technique could be a useful tool in placenta tissue-based studies when only cryo-compatible antibodies are available. Such antibodies can be used on cryostat sections only, giving results of considerably inferior morphological detail as compared to routinely fixed paraffin embedded tissue sections. Commercially available, only cryo-compatible, monoclonal antibodies against a conformational epitope of HLA-G (clone MEM-G/9) and leukocyte differentiation antigens CD56, CD163 and CD34 III were selected and applied to frozen sections, routinely formalin-fixed and HOPE-fixed paraffin sections. All tested antibodies immunolocalized their antigen on cryo sections and on HOPE-fixed but not formalin-fixed paraffin sections. The HOPE technique provides an excellent preservation of protein antigenicity together with well presented morphological details in paraffin embedded placenta tissues. The detection of native or conformation-dependent epitopes in paraffin sections expands the immunolocalization possibilities in placenta research and reproductive immunology.     

A technique for retrieving antigens in formalin-fixed, routinely acid-decalcified, celloidin-embedded human temporal bone sections for immunohistochemistry.

The application of immunohistochemistry to routinely decalcified, celloidin-embedded human temporal bone sections has been hampered because of antigen loss during processing of the specimens. To our knowledge, there has been no published report to date describing immunohistochemical staining of such tissues suitable for examination by light microscopy. Here we report a novel antigen retrieval technique which can be successfully used to stain a variety of antigens in routinely formalin-fixed, trichloroacetic acid-decalcified, celloidin-embedded human temporal bone sections. The new procedure reported here for decalcified human temporal bone tissues simply requires immersing slides for 30 min at room temperature in an antigen retrieval solution. A total of 60 decalcified, celloidin-embedded human temporal bone tissues were tested with monoclonal antibodies (MAb) to 15 different antigens. Of these, 12 MAb showed definite positive staining, while three were negative. This technique may prove very useful in studying the expression of various antigens by immunohistochemistry in formalin-fixed, acid-decalcified, celloidin-embedded tissues.     

Immunohistology on formol-sublimate fixed and paraplast embedded kidney tissue with comparison to formalin fixation and to pre-embedding immunofluorescent staining

Many alternative methods for immunopathological evaluation of kidney tissue are now available. Immunofluorescent or immunoperoxidase staining of kidney can be performed after formalin fixation and paraffin embedding. This is also possible after fixation with formol-sublimate (Stieve's fluid) using the immunoperoxidase technique or by immunofluorescence after removal of mercury. Reduction of strong nonspecific fluorescence caused by the mercury fixative parallels the elimination of mercury as verified by X-ray microanalysis of the sections. Using a mouse model with injection of graded dilutions of antiglomerular basement membrane antibodies, immunofluorescent staining after Stieve fixation and embedding in Paraplast was about 60% of that in cryostat sections. Immunofluorescent staining after mercury removal can be followed by silver staining for detailed morphologic study of the same 1 micron Paraplast sections. A case of antiglomerular basement membrane glomerulonephritis is illustrated in more detail to show the necessity of alternative methods, including the technique presented, pre-embedding immunofluorescent staining of Epon sections, and electron microscopy, to make a reliable diagnosis of this disease.     

Immunohistology on Formol-Sublimate Fixed and Paraplast Embedded Kidney Tissue with Comparison to Formalin Fixation and to Pre-Embedding Immunofluorescent Staining

Many alternative methods for immunopathological evaluation of kidney tissue are now available. Immunofluorescent or immunoperoxidase staining of kidney can be performed after formalin fixation and paraffin embedding. This is also possible after fixation with formol-sublimate (Stieve's fluid) using the immunoperoxidase technique or by immunofluorescence after removal of mercury. Reduction of strong nonspecific fluorescence caused by the mercury fixative parallels the elimination of mercury as verified by X-ray microanalysis of the sections. Using a mouse model with injection of graded dilutions of antiglomerular basement membrane antibodies, immunofluorescent staining after Stieve fixation and embedding in Paraplast was about 60% of that in cryostat sections. Immunofluorescent staining after mercury removal can be followed by silver staining for detailed morphologic study of the same 1 μm Paraplast sections. A case of antiglomerular basement membrane glomerulonephritis is illustrated in more detail to show the necessity of alternative methods, including the technique presented, pre-embedding immunofluorescent staining of Epon sections, and electron microscopy, to make a reliable diagnosis of this disease.     

Combined immuno- and non-radioactive hybridocytochemistry on cells and tissue sections: influence of fixation, enzyme pre-treatment, and choice of chromogen on detection of antigen and DNA sequences

Conditions for combination of DNA in situ hybridization, using biotinylated DNA probes, with immunohistochemistry were investigated on cryostat sections, cytological preparations, and paraffin sections. We found that cryostat sections and cytological preparations are suitable for in situ hybridization of target DNA after fixation in acetone, methanol, ethanol, or Carnoy without further proteinase pretreatment. Acetone is also very suitable for immunostaining of cell surface or cytoskeleton antigens. We therefore performed combined immunoenzyme and in situ hybridization staining using this fixative. The best results were obtained when immunoperoxidase staining with diaminobenzidine/H2O2 was followed directly by in situ hybridization. In addition to immunoperoxidase, alkaline phosphatase-antialkaline phosphatase (APAAP) staining with naphthol ASBI phosphate and New Fuchsin as a substrate could be used. In most instances, detection of the biotinylated hybrid with a streptavidin-biotinylated polyalkaline phosphatase method using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate as the substrate was preferable. The double stainings were studied on the following test models: (a) frozen tonsil sections: cell surface antigens (pan T) and ribosomal DNA; (b) frozen genital condyloma sections; cytokeratins and human papillomavirus type 6 + 11 (HPV-6/11) DNA; (c) CaSKi cells: cytokeratins and HPV-16 DNA; (d) infected fetal lung fibroblasts: vimentin and cytomegalovirus (CMV) DNA. An adapted procedure was followed on routinely formaldehye-fixed and paraffin-embedded condyloma tissue. Immunoperoxidase staining for papilloma virus capsid antigen could be combined with DNA in situ hybridization with HPV-6/11 DNA. In this model, however, the accessibility of the target DNA had to be improved by enzyme treatment after the immunostaining and before starting the in situ hybridization.     

Frozen Sections from Bouin-Fixed Material in Histopathology

It has become almost a dogma that, for obtaining satisfactory frozen sections, fixation of the tissues in formalin is necessary; formalin is exclusively recommended for this purpose in the textbooks of microscopical and pathological technic.¹ It has, however, several important disadvantages, which become the more acutely felt when fixation in more than one fixative is impossible, as happens so often in surgical pathology. When, after the preparation of the frozen sections, embedding in paraffin is necessary, formalin-fixed tissues show a marked shrinkage, especially when rapid embedding methods (e. g. dioxane) are used. This shrinkage can only be prevented by the time-consuming hardening of the blocks in K₂Cr₂O₇. The trichrome stains, the phosphotungstic-hematoxylin stain and the azan method, which are slowly superseding the hematoxylin-eosin and van Gieson stains in histopathology, give only mediocre or inferior results after formalin fixation, unless the sections are refixed.     

Lymphocyte membrane antigens in glycol methacrylate embedded tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

Lymphocyte Membrane Antigens in Glycol Methacrylate Embedded Tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

Immunofluorescent Labelling of K-Papovavirus Antigens in Glycol Methacrylate Embedded Material: A Method for Studying Infected Cell Populations by Fluorescence Microscopy and Histological Staining of Adjacent Sections

Trypsin and protease V (pronase) were studied for their ability to enhance immuno-fluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 μm sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

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.     

Optimization of immunohistochemical techniques to detect extracellular matrix proteins in fixed skin specimens

Complete antigen visualization in the context of well-preserved tissue architecture is the goal of all immunohistochemical techniques. Frozen tissue section techniques achieve optimal antigen visualization but preserve tissue architecture poorly. On the other hand, formalin-fixed tissue section techniques preserve tissue architecture very well but result in antigen masking. Enzymatic digestion or salt extraction of formalin-fixed sections has been used to reestablish antigen expression. Recently acid-alcohol-fixed tissue has been used as a successful compromise between tissue architecture preservation and the visualization of cytoskeletal antigens. In an attempt to find an improved immunohistochemical process for non-cytoskeletal antigens, we compared avidin-biotin immunofluorescence staining in frozen, formalin-fixed, and acid-alcohol-fixed tissues. The fixed tissues were either untreated or treated with enzyme digestion or salt extraction. For this study, we examined healing cutaneous wounds in Yorkshire pigs with antibodies to fibronectin, laminin, von Willebrand factor VIII, and keratin. Although tissue architecture was poor, frozen sections provided the best antigen visualization and were therefore used as the standard for complete antigen expression. Formalin-fixed tissues had excellent tissue architecture, but most antigens were completely masked. Pre-treatment technique only partially overcame the antigen masking caused by formalin. In contrast, acid-alcohol fixation preserved tissue architecture almost as well as formalin and sometimes allowed complete antigen visualization; however, laminin and fibronectin were partially masked. Total recovery of the expression of these antigens could be obtained by pre-treating the acid-alcohol-fixed tissue with either hyaluronidase or 1 M NaCl. Therefore, acid-alcohol-fixed tissue appears best for extracellular matrix (ECM) protein immunostaining as well as for cytoskeletal staining. However, certain ECM antigens require hyaluronidase or 1 M NaCl treatment for optimal visualization.     

Immunofluorescent labelling of K-papovavirus antigens in glycol methacrylate embedded material: a method for studying infected cell populations by fluorescence microscopy and histological staining of adjacent sections

Trypsin and protease V (pronase) were studied for their ability to enhance immunofluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 micron sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

Reversing the effects of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method.

Formalin is a commonly used fixative for tissue preservation in pathology laboratories. A major adverse effect of this fixative is the concealing of tissue antigens by protein cross-linking. To achieve a universal antigen retrieval method for immunohistochemistry under a constant condition, we developed a new method in which the effects of formalin fixation were reversed with citraconic anhydride (a reversible protein cross-linking agent) plus heating. Formalin-fixed, paraffin-embedded tissues from various organs were examined for immunohistochemical localization of a wide variety of antigens. Deparaffinized tissue sections were placed in an electric kitchen pot containing 0.05% citraconic anhydride solution, pH 7.4, and the pot was set at "keep warm" temperature mode of 98C for 45 min. This mode allowed heating the sections at a constant temperature. The sections were then washed in buffer solution and immunostained using a labeled streptavidin-biotin method using an automated stainer. In general, formalin-fixed tissues demonstrated specific immunostainings comparable to that in fresh frozen tissues and significantly more enhanced than after conventional antigen retrieval methods. In particular, even difficult-to-detect antigens such as CD4, cyclin D1, granzyme beta, bcl-6, CD25, and lambda chain revealed distinct immunostainings. Different classes of antigens such as cellular markers and receptors, as well as cytoplasmic and nuclear proteins, consistently produced enhanced reactions. This method provides efficient antigen retrieval for successful immunostaining of a wide variety of antigens under an optimized condition. It also allows standardization of immunohistochemistry for formalin-fixed tissues in pathology laboratories, eliminating inter-laboratory discrepancies in results for accurate clinical and research studies.     

Formalin is deleterious to cytoskeleton proteins: do we need to replace it by formalin-free Kryofix?

Formalin is hazardous for the environment and for the laboratory personnel and deleterious to cytoskeleton proteins. The pathology and anatomy laboratory can be formalin-free when Kryofix is used as a substitute fixative. In four years experience with Kryofix, we learned that immunostaining on paraffin sections is reproducible and of excellent quality. The current study concerns the comparison of interfilament staining on paraffin sections made from formalin or from Kryofix-treated tumors. For both keratin and vimentin there were false-negative findings in the formalin-fixed tumors. Of these two, vimentin was het most susceptible to formalin fixation. Three of the ten adenocarcinomas were positive for both keratin and vimentin, indicating that this double staining pattern is not uncommon. Postfixation of Kryofix-treated tissue with formalin results in false-negative immunostaining.     

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

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

Differential sensitivity of the microtubule-associated protein, tau, in Alzheimer's disease tissue to formalin fixation

Immunohistochemistry of formalin-fixed human Alzheimer's disease (AD) tissue using an anti-tau antibody (Tau-1) reveals staining of neurofibrillary tangles (NFTs) and neuritic plaques (NPs), whereas normal axonal staining is less apparent. In this study, we used a combined biochemical and histochemical approach to assess effects of formalin on immunoreactivity of AD tau. Nitrocellulose blots were treated with fixative to mimic conditions used with tissue sections, a method that might be generally useful for assessing antigen sensitivity to different fixatives. A progressive decrease in Tau-1 immunoreactivity of the tau bands on a Western blot was observed with increasing times of formalin fixation. Phosphatase-digested blots demonstrated an increase in Tau-1 immunoreactivity compared to control blots. These results mimic the phosphatase-sensitive Tau-1 immunohistochemical staining of formalin-fixed AD tissue slices previously reported. Fixation of AD tissue with periodate-lysine-paraformaldehyde (PLP) preserves axonal tau antigenicity. Phosphatase digestion of PLP-fixed AD tissue enhances Tau-1 immunoreactivity of NFTs and NPs but does not alter axonal staining. These results indicate that axonal form(s) of tau are more sensitive to formalin fixation than pathology-associated tau. In addition, a modification of AD tau in pathological structures may protect it from the effects of formalin with regard to Tau-1 antigenicity.     

Variability of laminin immunoreactivity in human autopsy brain.

Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.     

An improved timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues

Summary Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described.To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixative is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

Comparative usefulness of tissue fixatives for in situ viral nucleic acid hybridization

Traditionally tissues for in situ hybridization of viral nucleic acid have been small pieces obtained from laboratory rodents, and fixatives that are designed for electron microscopy, such as periodate-lysine-paraformaldehyde (PLP) can handle them adequately. However, these fixatives have limited penetrating ability and may produce no appreciable hardening, so alternative fixation methods were evaluated. The intention was to determine whether fixatives adequate for bulky tissues such as whole or halved pig and cow brains would also be compatible with in situ hybridization. Various fixatives were evaluated using a system of intracranial inoculation of BALB/c mice with pseudorabies virus (PRV) followed by in situ hybridization of brain tissue sections with a 35S-labeled PRV DNA probe. Loss of tissue sections was a major problem, particularly with PLP and formalin, but positive results were obtained with five fixatives tested. Cellular morphology was especially good with PLP and with a modification of Carnoy's fluid, MOCA fixative. An incidental but important observation was that formalin is compatible with in situ hybridization. Retroactive studies of viral diseases using routinely processed blocks of tissue (formalin-fixed, paraffin-embedded) are therefore conceivable.