Fixation-dependent cytoplasmic false-positive staining with an immunoperoxidase method

Fixation-dependent nonspecific staining with the unlabeled immunoperoxidase (PAP) method was studied using paraffin-embedded human spleen sections fixed in various fixatives; the specific primary antiserum was omitted or nonimmunized normal rabbit serum was used. Strong cytoplasmic staining of polymorphonuclear leucocytes and macrophages was found after fixation in acetone, alcoholic formalin (94% alcohol) and absolute ethanol. This staining was mainly produced by the second layer of the PAP method. The most probable explanation of this phenomenon is nonspecific protein-immunoglobulin interaction as a result of alcoholic or acetone fixation of the sections. The present findings point to the importance of controls for each case under study to avoid false-positive interpretations.     

Sensitivity and nonspecific staining of various immunoperoxidase techniques

Summary Optimally fixed paraffin embedded tissue sections and cytocentrifuged cell smears were used to test the sensitivity and nonspecific staining with the enzyme-bridge, PAP, indirect and direct immunoperoxidase methods using human immunoglobulins and lysozyme as antigens. With the enzyme-bridge method positive staining was seen with primary antiserum dilutions up to 1:20,000. The least background staining was observed with this method. The PAP method was equally sensitive, although false-negative results with low primary antiserum dilutions were seen. Some nonspecific background staining always persisted using the PAP method even with high primary antiserum dilutions. The indirect method was not as sensitive as the enzyme-bridge method and some nonspecific staining always persisted. The direct method was too insensitive with paraffin embedded tissue sections.Supported by the Sigrid Jusélius Foundation and Finska Läkaresällskapet     

Demonstration of myoglobin and CK-M in myocardium. Comparison of five fixation methods and three immunohistochemical techniques

The results of immunohistochemical staining vary depending on the tissue, fixative, antigen-antibody system, and immunohistochemical staining methods used. The purpose of this study was to evaluate the effect of different methods of fixation, different antigen-antibody systems, and different immunohistochemical methods on immunohistochemical staining of myocardium. Samples of normal fresh canine myocardium from six dogs were fresh frozen and fixed in 10% neutral buffered formalin, Bouin's, Bayley's and Carnoy's fixatives. Immunohistochemical staining for myoglobin and creatine kinase M was performed using the ABC (avidin-biotin complex) and indirect peroxidase-antiperoxidase (PAP) techniques. Tissues fixed in formalin showed the most intense specific staining for both antigens with the least background and nonspecific staining. All other fixation methods and frozen section techniques gave a more variable degree of specific positive staining and substantial background staining and/or nonspecific staining. ABC and PAP techniques gave similar results with both antigen-antibody systems and with each fixation method. Thus, no differences in specificity or sensitivity were observed between ABC and PAP techniques. Differences in staining intensity and pattern were related primarily to differences in fixation methods.     

Sensitivity and nonspeicific staining of various immunoperoxidase techniques

Optimally fixed paraffin enbedded tissue sections and cytocentrifuged cell smears were used to test the sensitivity and nonspecific staining with the enzyme-bridge, PAP, indirect and direct immunoperoxidase methods using human immunoglobulins and lysozyme as antigens. With the enzyme-bridge method positive staining was seen with primary antiserum dilutions up to 1:20,000. The least background staining was observed with this method. The PAP method was equally sensitive, although false-negative results with low primary antiserum dilutions were seen. Some nonspecific background staining always persisted using the PAP method even with high primary antiserum dilutions. The indirect method was not as sensitive as the enzyme-bridge method and some nonspecific staining always persisted. The direct method was too insensitive with paraffin embedded tissue sections.     

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.     

Influence of fixatives on the immunoreactivity of paraffin sections

Summary The unlabelled antibody peroxidase-antiperoxidase (PAP) technique was used to demonstrate IgA in paraffin sections of human tonsil fixed in eight commonly known fixatives. In all but one of the fixatives, treatment with trypsin prior to immunostaining resulted either in digestion of sections, or no change in positive staining. In tissues fixed in neutral buffered formalin-saline, positive staining was absent unless the sections had been treated with trypsin, and it has been shown that digestion requirements of different areas throughout a section may vary according to the effectiveness of fixation.     

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.     

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.     

Precautions in the application of immunohistochemical techniques to tissues of the pig hypothalamo-neurohypophysial system.

Immunohistochemical procedures were used to localize neurophysin in the hypothalamo-neurohypophysial axis of the domestic pig. The topographical distribution of neurophysin as revealed by the immunofluorescence "sandwich" technique was similar to that found when either the immunoglobulin-peroxidase bridge method or the peroxidase-labeled gamma-globulin technique was employed. However, application of the peroxidase-anti-peroxidase (PAP) complex procedure resulted in nonspecific staining of the magnocellular structures. This phenomenon was attributed to the action of PAP on the tissue and after screening a number of other vertebrate species was found to be unique to the pig. Minimal nonspecific binding of the PAP could be achieved either by reducing the reaction time of PAP to 5 min or, by the addition of 1% (v/v) normal serum to all reagents and wash solution. That the PAP-binding protein is a component of the hypothalamo-neurohypophysial axons is discussed.     

A new fixed cryosection technique for the simultaneous immunocytochemical demonstration of T6 and S100 antigens

Summary A formalin—calcium fixation method of preparing cryosections is described, which allows demonstration of Langerhans' cells by S100 antigen staining on frozen sections. The number of Langerhans' cells given by T6 antigen staining is also higher in formalin—calcium fixed frozen sections than acetone fixed frozen sections. The preparation is suitable for dual demonstration of the two antigens on the same section enabling a more accurate numerical evaluation of Langerhans' cell populations in the normal cervical epithelium.     

Improvements in histological techniques for epoxy-resin embedded bone specimens

A procedure is presented in which some of the processing difficulties with fixation, embedding and cutting whole mouse bones and large bone pieces from other species are considered. The bone specimens are fixed in acetone or by a Karnovsky-formol-saline process which preserves intact endosteal surface-to-cortex layers. After fixation the bones are embedded in a hard mixture of epoxy resin to provide blocks with face sizes up to 3.5 x 3.0 cm. Mineralized sections are cut to 4 micrometer; demineralized at 3 micrometer. Sections are fastened to gelatin-subbed slides with pressure plates which produce flat, secure sections. After removal of the plastic, an unmodified Mayer's hematoxylin and a polychromatic eosin staining method is applied to demineralized sections, and a slightly modified method to mineralized sections.     

Improvements in Histological Techniques for Epoxy-Resin Embedded Bone Specimens

A procedure is presented in which some of the processing difficulties with fixation, embedding and cutting whole mouse bones and large bone pieces from other species are considered. The bone specimens are fixed in acetone or by a Karnovsky-formol-saline process which preserves intact endosteal surface-to-cortex layers. After fixation the bones are embedded in a hard mixture of epoxy resin to provide blocks with face sizes up to 3.5 × 3.0 cm. Mineralized sections are cut at 4 μm; demineralized at 3 μm. Sections are fastened to gelatin-subbed slides with pressure plates which produce flat, secure sections. After removal of the plastic, an unmodified Mayer's hematoxylin and a polychromatic eosin staining method is applied to demineralized sections, and a slightly modified method to mineralized sections.     

Immunocytochemical staining of cytocentrifuge prepared cultured cells: nonspecific staining and its elimination

Summary In an attempt to localize hormones in cytocentrifuge-prepared cultured cells of small cell carcinoma of the lung (SCCL), various modifications of the immunoperoxidase (PAP) procedure (Sternberger, 1979) were tested. When using glutaraldehyde, formaldehyde, orp-benzoquinone fixation (Pearse & Polak, 1975) and rabbit antibodies in primary or bridging steps of the PAP procedure, nonspecific staining (false positives) could be elicited with the majority of rabbit antibodies tested, but not with antibodies from other animal sources. This problem could be eliminated by fixation of cells either with formalin-acetone (Masonet al., 1975) or, when using antibodies from a source other than rabbit, glutaraldehyde. It was not possible to localize ACTH in DMS-79, a human SCCL line known to produce this hormone. However, calcitonin was localized in the calcitonin-producing SCCL line DMS-53. Failure to localize ACTH in DMS-79 may be due to the lower levels of this hormone in DMS-79, as compared to the levels of calcitonin in DMS-53. This study emphasizes the importance of proper controls before concluding successful localization in a given immunocytochemical preparation of cultured cells.     

Comparison between peroxidase-conjugated antigen or antibody and peroxidase-anti-peroxidase complex in a postembedding procedure.

The staining efficiency of peroxidase labeled immunoglobulin conjugate, used either as antigen or as antibody, has been compared with that of peroxidase-anti-peroxidase complex (PAP) on ultrathin sections of araldite embedded material. The conjugate gave positive results in a two layer method as well as in a three layer method when used as antibody. No staining was observed when it was used as antigen. The conjugation seemed to impair the antigenic reactivity of immunoglobulin. The conjugate when used as antibody in the three layer method gave approximately the same staining efficiency as PAP.     

Modified silver staining of nucleolar organizer regions to improve the accuracy of image analysis

Silver staining of nucleolar organizer regions (NORs) and their subsequent quantification by image analysis are used increasingly in human pathological specimens and experimental models. Because certain conditions determined by the type of tissue and/or its fixation render AgNOR segmentation for image analysis difficult due to insufficient contrast or nonspecific silver precipitation, we propose three improvements to the original technique to overcome these difficulties. Pretreatment with 7% nitric acid produced very distinct dark brown images of AgNORs on a yellow background. The gradient of background colors allowed easy discrimination of nucleolar, nuclear and cytoplasmic structures. Seven morphometric parameters related to number, size and shape of AgNORs were evaluated quantitatively by image analysis on sections pretreated with nitric acid and on adjacent sections treated with citrate buffer in a wet autoclave according to the most widely accepted method for image analysis of AgNOR. Both methods yielded similar results. A second improvement was achieved by coating the slides with 7% celloidin solution in ethyl alcohol-ether prior to AgNOR staining and acid pretreatment. This coating prevented nonspecific silver deposition on argyrophilic bacteria and other tissue debris in human vaginal smears that could make visualizing AgNOR sites difficult. Finally, placing sections face down on the staining solution prevents the formation of nonspecific silver precipitates. These procedures can be applied together or separately according to the requirements of the material to be evaluated.     

Histological Technic for Cerebellar Climbing and Mossy Fibers

In order to, avoid disadvantages attendant upon the use of fresh frozen sections, or of block impregnation with silver, in staining climbing or mossy fibers of the cerebellum, Rio Hortega's double impregnation method for nerve fibers is useful. This consists of prolonged formalin fixation prior to cutting frozen sections (which thereafter are easier to cut) and preliminary treatment with ammoniacal aqueous and alcoholic washes, mordanting in pyridine silver, and treatment with pyridine-silver-carbonate. Following this, sections are handled individually through one of several reduction methods after which they may be directly mounted or gold toned.     

Modified silver staining of nucleolar organizer regions to improve the accuracy of image analysis

Silver staining of nucleolar organizer regions (NORs) and their subsequent quantification by image analysis are used increasingly in human pathological specimens and experimental models. Because certain conditions determined by the type of tissue and/or its fixation render AgNOR segmentation for image analysis difficult due to insufficient contrast or nonspecific silver precipitation, we propose three improvements to the original technique to overcome these difficulties. Pretreatment with 7% nitric acid produced very distinct dark brown images of AgNORs on a yellow background. The gradient of background colors allowed easy discrimination of nucleolar, nuclear and cytoplasmic structures. Seven morphometric parameters related to number, size and shape of AgNORs were evaluated quantitatively by image analysis on sections pretreated with nitric acid and on adjacent sections treated with citrate buffer in a wet autoclave according to the most widely accepted method for image analysis of AgNOR. Both methods yielded similar results. A second improvement was achieved by coating the slides with 7% celloidin solution in ethyl alcohol-ether prior to AgNOR staining and acid pretreatment. This coating prevented nonspecific silver deposition on argyrophilic bacteria and other tissue debris in human vaginal smears that could make visualizing AgNOR sites difficult. Finally, placing sections face down on the staining solution prevents the formation of nonspecific silver precipitates. These procedures can be applied together or separately according to the requirements of the material to be evaluated.     

Immunofluorescent staining of histaminase (diamine oxidase) in human placenta.

An immunofluorescent procedure for the localization of histaminase in human tissue sections has been developed by using a specific antiserum against human placental histaminase. For localization of this enzyme in placental sections, fixation in equal volumes mixture of absolute ethanol and acetone provided the optimum visualization of this enzyme in both frozen sections and paraffin-embedded sections. The immunofluorescent staining of this enzyme in placenta is found to be localized in areas within the maternal decidua, both within the cytoplasm of the decidual cells and in tissue space between the cells. The chorionic villi are completely void of the immunofluorescent stain. Variations in patterns of histaminase localization have been found between term and premature placentas, with the former showing a predominantly intercellular localization and the latter a predominantly intracellular localization. The intercellular localization of this enzyme in the decidua may represent a nonspecific diffusion of the enzyme associated with delivery of the placenta or may reflex a specific functional role of the enzyme in the intercellular space during pregnancy.     

Methods for decreasing interstitial immunoglobulin in tissue slices and cryostat sections

To determine whether lymphoid antigens and cellular morphology can be preserved after long-distance transport in buffer or cell culture medium, we stained cryostat sections prepared from human tonsil samples that had been kept at 4 degrees C or 20 degrees C for 24, 48 or 72 h. B-Cell antigens, T-cell antigens, and Ia antigens were well preserved after storage up to 72 h in buffer or medium at 4 degrees C. Interstitial immunoglobulin (Ig) was decreased following all incubation procedures. We then investigated methods to diminish interstitial Ig in cryostat sections, since it would be inconvenient to keep 2-3 mm tissue slices in buffer or medium prior to freezing and subsequent Ig staining. Cryostat sections were air dried or briefly fixed in acetone prior to washing in buffer or medium at 4 degrees C, 20 degrees C or 37 degrees C for 1, 2 or 24 h. Then sections were air dried or washed prior to acetone fixation and immunostaining. A method for washing cryostat sections was developed which diminished interstitial Ig without compromising the quality of immunostaining or cellular detail. These methods are especially useful for studying samples of lymphoid tissue in which the presence of large quantities of interstitial Ig obscures the detection of monotypic Ig staining patterns.