Immunohistochemical localization of human immunoglobulins and lysozyme in epoxy-embedded lymph nodes: effect of different fixatives and of proteolytic digestion

The postembedding immunoperoxidase staining technique for the localization of immunoglobulins (light and heavy chains) and of lysozyme has been successfully applied to epoxy-embedded human lymph nodes, after removal of the resin. Glutaraldehyde-containing fixatives appear to be suitable for the immunohistochemical localization of human immunoglobulins and lysozyme, provided that the masked antigenicity of these proteins is recovered by proteolytic digestion of the tissue sections using 0.4% pepsin or 0.1% trypsin. Nonglutaraldehyde-containing fixatives allow the immunolocalization of human immunoglobulins without any enzymatic pretreatment. This study shows that tissues routinely fixed in glutaraldehyde and embedded for ultrastructural investigations are actually suitable for immunohistochemical studies on human immunoglobulins and lysozyme.     

Production of antibodies specific for double stranded antigen DNA cloned from immune complexes in plasma of a SLE patient.

The antigenicity of antigen DNA isolated from immune complexes in plasma of a SLE patient was examined. DDY mice were immunized with the cloned antigen DNA carrying a sequence homologous with a part of bacteriophage f1 (KS8 DNA) by the coupling method, and the antibody response was estimated by radioimmunoassay. Antibodies specific to double stranded DNA were elicited. Moreover, the antibodies showed preferential binding to KS8 DNA than other DNA derived from Escherichia coli. These results suggest that KS8 DNA has a significant antigenicity in mice.     

Processing techniques for the demonstration of myelin basic protein in paraffin-embedded optic nerve: an immunoperoxidase study of the developing albino rat

A comparison was made of the effects of various fixation and processing conditions upon the antigenicity of myelin basic protein (MBP) in sections of paraffin-embedded optic nerve from the developing albino rat as judged by the unlabeled peroxidase-antiperoxidase technique. The fixatives used were: Perfix, 4% and 2% buffered paraformaldehyde (pH 7.4), 10% buffered formalin (pH 7.4); Bouin's, Clark's, and Carnoy's fixatives, and 20% formalin in a solution of HgCl2 that had been saturated at 1 degrees C. Perfix appeared to be the best fixative for the preservation of morphology and MBP antigenicity during the early stages of myelinogenesis but was not satisfactory during the later stages. The buffered aldehydes were slightly more destructive of MBP antigenicity than was Perfix, but they produced satisfactory results following the first postnatal week. Bouin's fixative was similar in effect to the buffered aldehydes, but nonspecific background staining was higher. HgCl2/formalin, Clark's and Carnoy's fixatives were unsuitable. No differences were noted in staining between material processed for embedding using 5, 30, or 60 min schedules.     

Electrophoretic protein blots as aids in choosing fixatives for immunocytochemistry

We used electrophoretic protein blots prepared from polyacrylamide gels to test the effect of different fixatives on the antigenicity of rough endoplasmic reticulum (RER) peptides from rat liver. Protein blots were prepared by the procedure of Towbin et al. (Proc Natl Acad Sci USA 76:4350, 1979), treated with different fixatives, rinsed to inactivate non-specific reactive sites, and then reacted with rabbit polyclonal anti-rat liver RER antibodies, followed by peroxidase-conjugated anti-rabbit antibodies. On the basis of differences in immunostaining densities as determined by densitometry, we found that RER peptides displayed differential sensitivities to various fixatives. Anti-rat liver RER antibodies and the immunogold technique were applied to methacrylate sections of in vitro fixed rat liver rough microsomes. Specific labeling was observed over the microsomes and was shown by quantitation to vary in a similar manner to the immunostaining of specific peptides in protein blots following different fixations. We conclude that protein blots may serve as useful tools for screening the effects of different fixatives on cell antigenicity, and therefore may be helpful in immunocytochemical studies.     

Immunocytochemical demonstration of vasopressin binding in rat kidney

We investigated the immunoperoxidase demonstration of vasopressin (VSP) bound to paraffin-embedded sections of rat kidney and the effects of various fixatives. Slices of rat kidney from normal and 4-day water-deprived rats were incubated with 10(-7) M VSP, fixed, and embedded in paraffin. Hydrated sections of these tissues were again incubated with 10(-7) M VSP or 10(-7) M VSP and 10(-5) M oxytocin (OXY). VSP bound to the sections was demonstrated using rabbit anti-Arg8 VSP antiserum and peroxidase-labeled second antibody. In sections of kidney from both normal and water-deprived rats, immunoperoxidase labeling was most intense in the renal papilla and was restricted to the cells of the ducts of Bellini and loops of Henle. In the medulla, the collecting ducts and medullary thick ascending limbs of Henle were moderately stained. In the normal kidney sections there was no staining of the proximal tubules, distal convoluted tubules (DCT), and only slight staining of the cortical collecting ducts (CCD). However, in the water-deprived rats there was a considerable increase in the staining of the DCT and CCD. Simultaneous incubation in OXY and VSP resulted in reduced immunoperoxidase labeling of the tubules. Omission of VSP incubation led to a similar decrease in stain intensity, indicating a specificity for the sites of VSP binding. This technique allows the identification of cells responsible for the binding of VSP in the kidney.     

Evaluation of nine different fixatives. 2. Preservation of IgG, IgA and secretory component in an artificial immunohistochemical test substrate

An artificial substrate was developed for quantitative testing of the ability of various fixatives to preserve the reactivity of IgG and IgA isotypes (gamma and alpha chains) and the secretory component (SC) of secretory IgA as model antigens. Polymerized normal rabbit serum was used as matrix and defined amounts (10-0.1 g/l) of antigen were incorporated into it by diffusion before fixation and paraffin embedding. The various fixatives comprised alcohol, routine formalin, glutaraldehyde(1%)-formalin, Baker's formol calcium, formol sublimate, acetic acid(2%)-formol saline, Bouin's fluid, Susa fixative, and carbodiimide. The detection sensitivity afforded by these fixatives was defined as the immunofluorescence staining end point. Compared to the reference value obtained with alcohol (gamma and alpha chains, 0.06 g/l of IgG and IgA; SC, 0.12 g/l of colostral IgA), an antigen concentration at least 8 times higher was necessary for detection with most of the cross-linking fixatives. Bouin's and Susa fixatives were peculiar in that they required more than 150 times higher antigen concentration for detection of IgG but only 3-8 times higher for IgA. The determined sensitivities were compared with the immunofluorescence performance results obtained on human tissues prepared with the same fixatives; excepting carbodiimide (which produced unacceptable autofluorescence of the substrate matrix) a remarkably good correlation was found with regard to IgG- and IgA-producing cells (especially of the former isotype) and secretory epithelium (IgA and SC). However, the latter result depended on pronase treatment of the tissue sections to unmask epithelial antigens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ia antigens in plastic-embedded tissues: a post-embedding immunohistochemical study

The aim of the present study was to establish a plastic embedding technique that makes possible the immunohistochemical demonstration of class II major histocompatibility complex (MHC) antigens (Ia antigens) in undecalcified joint tissues. Therefore a series of fixatives and dehydrating agents was tested for saving Ia immunoreactivity by post-embedding immunostaining of thin sections (2 microns) of rat tissues that had been embedded in glycol methacrylate (GMA), and by comparing with cryostat sections. An indirect immunoperoxidase and the avidin-biotin complex (ABC) technique were used. Combined with fixation by 4% formaldehyde, dehydration with GMA was found to give the best preservation of Ia antigenicity, followed by dehydration with ethylene glycol. The thinness of tissue sections facilitated the association of Ia antigens with different subcellular compartments in distinct cell populations. These various patterns are described.     

Human carbonic anhydrase isoenzyme C

Summary The effects of some alcohol and aldehyde containing fixatives on the antigenicity of human carbonic anhydrase isoenzyme C (HCA C) were tested in order to reveal the most suitable method for the immunohistochemical localization of this enzyme. The use of 2% and 4% paraformaldehyde or 2% glutaraldehyde solutions before immunoperoxidase (PAP) staining resulted in the loss of HCA C-specific reactivity in the surface epithelial cells of human appendicular and gastric mucosae, whereas the antigenic reactivity of HCA C was well retained in the parietal cells of gastric glands. In corresponding tissue sections fixed with one of the alcohol containing solutions (abs. methanol, methanol+chloroform 21 or Carnoy fluid) both the surface epithelial and parietal cells showed HCA C-specific immunostaining after the PAP procedure. In addition, the antigenicity of HCA C was found to be well preserved in some tubular cells of human kidney fixed in Carnoy fluid. The paraffin infiltration at relatively low temperature did not markedly affect the enzyme antigenicity. Fixation in Carnoy fluid coupled with paraffin embedding at 55–60° C in vacuo was found to give the best preservation of the antigenicity of HCA C with good morphological integrity for light microscopical localization.Grant support from the Finnish Culture Foundation     

Partial delipidation improves the T-cell antigenicity of hepatitis B virus surface antigen

Hepatitis B virus surface antigen (HBsAg) is a complex macromolecular particle composed of glycoproteins and lipids. The latter, representing 25% of the particle mass, are of host origin and determine the solubility, stability, and, indirectly, B-cell immunogenicity of HBsAg. HBsAg is a T-cell-dependent immunogen that does not elicit a detectable humoral immune response in 5% of HBsAg vaccine recipients and in most subjects suffering from chronic hepatitis B. We investigated the influence of the lipid content on the antigenicity of the particle. Lipids were partially removed from HBsAg by treatment with beta-D-octyl glucoside and density centrifugation. Sham treatment consisted of density centrifugation of HBsAg only. We compared the in vitro proliferative responses of established T-cell lines and nonfractionated peripheral blood mononuclear cells (PBMC) from HBsAg vaccinees and chronic HBV patients when stimulated with partially delipidated HBsAg, untreated HBsAg, or sham-treated HBsAg. In all experiments, delipidated HBsAg turned out to be 10 to 100 times more antigenic than its untreated or sham-treated counterpart. Remarkably, PBMC from vaccine nonresponders or chronic HBV patients displayed a proliferative response towards delipidated HBsAg, whereas native HBsAg never induced a response. A series of control experiments demonstrated that this enhancement of T-cell antigenicity was HBsAg specific and directly linked to lipid extraction. Nonspecific adjuvant effects of any kind could be ruled out. In vivo evaluation in mice demonstrated that delipidated particles lose most of their B-cell antigenicity. However, when native and delipidated particles were mixed, these mixtures induced equal or slightly superior anti-HBs responses to those induced by the same quantity of native HBsAg alone. In conclusion, our data show that partial delipidation of HBsAg strikingly increases the T-cell antigenicity of this unique viral antigen.     

BCG-CpG-DNA对重组HBsAg免疫原性的影响

为了研究BCG-CpG-DNA对重组HBsAg免疫原性的影响,了解其佐剂功能。采用不同剂量BCG-CpG-DNA与不同剂量重组(汉逊酵母)表达的HBsAg或Al-HBsAg混合免疫小鼠,与同剂量铝佐剂疫苗对比,以放射免疫法检测抗-HBs中和抗体水平和抗体持续时间,ELISA法检测抗体亚类。结果显示,BCG-CpG-DNA和HBsAg混合,抗-HBs的中和抗体水平达到或高于同剂量铝佐剂疫苗;BCG-CpG-DNA和Al-HBsAg混合,抗-HBs的中和抗体水平高于同剂量铝佐剂疫苗,并有统计学显著意义(P<0.05);添加BCG-CpG-DNA组抗体水平4周时增加不明显,到10周则显著地高于同剂量铝佐剂疫苗组,IgG2a抗体水平也高于相应的对照组。实验结果表明BCG-CpG-DNA对HBsAg有较好的免疫佐剂作用,并和AL佐剂有协同作用。     

Hepatitis B virus surface antigen can activate dendritic cells and modulate T helper type immune response

Hepatitis B virus surface antigen (HBsAg) is a major antigen of hepatitis B virus (HBV). Dendritic cells (DC) of HBV carriers have been reported to exhibit functional impairment. In this study, the role of HBsAg on mice bone marrow-derived dendritic cells and immune responses in vivo was studied. The immune modulatory function of HBsAg was explored by using mice bone marrow-derived dendritic cells in vitro and also by examining an ovalbumin (OVA) specific immune response in vivo. Treatment of dendritic cells with HBsAg resulted in enhanced cell surface expression of cluster of differentiation (CD) 80, CD83, CD86, and major histocompatibility complex (MHC) class II, and enhanced production of interleukin (IL)-12 p40 and IL-12 p70. Treatment of dendritic cells with HBsAg resulted in decreased T cell secretion of IL-5 by OVA stimulation. In addition, the results showed stronger OVA-specific immunoglobulin (Ig) M and weaker IgG responses in mice sera when they had been immunized with OVA and co-injected with HBsAg. It was also found that the mice exhibited significant enhancement of anti-OVA IgG2a antibody (Ab), as well as marked inhibition of IgG1 Ab production. In cellular immune responses, IL-5 production was significantly decreased and interferon (IFN)-γ increased in the group co-injected with HBsAg. On the other hand, the induction of lymphoproliferative response to OVA stimulation in spleen cells was decreased in the HBsAg co-injected group. These results demonstrate that HBsAg can affect the differentiation of T helper (Th) cells, which might provide a strategy for improving its prophylactic and therapeutic efficacy.     

Evaluation of tissue preparation methods and paired immunofluorescence staining for immunocytochemistry of lymphomas

Eight cross-linking fixatives were tested for preservation of extracellular or intracellular IgG, IgA, IgM, IgD, kappa and lambda light chains, J chain and secretory component. Most of the selected fixatives have been used in recent immunohistochemical studies of lymphoproliferative processes and comprised routine formalin, glutaraldehyde(1%)-formalin, Baker's formalin-calcium, formalin-sublimate, acetic acid(2%)-formalin-saline, Bouin's fluid, Susa fixative, and carbodiimide. The results obtained in artificial test substrates with defined amounts of IgG or IgA and in biological substrates (colon mucosa, tonsils, and different types of lymphomas) were compared by immunofluorescence with the antigenic preservation afforded by fixation in cold 96% ethanol (with or without inclusion of a pre-fixation 48 h washing period). An antigen concentration at least an eight-fold higher was necessary for detection with most other fixatives. Bouin's and Susa fixatives were peculiar in that they required antigen concentration 150 times higher for detection of IgG but only 3-8 times higher for IgA. Light chains were relatively well preserved by all fixatives except glutaraldehyde. For all cross-linking fixatives, the extent of antigenic masking depended on the concentration of environmental proteins, and the efficiency of unmasking with pronase or trypsin, therefore, varied with the location in the tissue. The J chain was particularly vulnerable to degradation during proteolytic treatment. The extensive masking of extracellular immunoglobulin in formalin-fixed tissue afforded a relatively good signal-to-noise ratio for immunoglobulin-producing cells when kappa and lambda chains were traced. Thus, differentiation between polyclonal and monoclonal B-cell processes on the basis of cytoplasmic labelling was often better in undigested sections. However, the light-chain type of membrane immunoglobulin could usually not be determined in directly fixed tissue. Ethanol fixation preceded by washing in saline afforded such determination and also preserved certain T-cell and HLA-DR antigens as well as diffuse alpha-naphthylbutyrate esterase. Reactive and malignant macrophages could further be traced by their cytoplasmic expression of L1 antigen, both in formalin- and ethanol-fixed material.     

Immunolocalization of manganese superoxide dismutase in normal and transgenic mice expressing the human enzyme

Summary The localization of manganese superoxide dismutase (MnSOD) was determined using immunohistochemistry of various tissues of normal and transgenic mice which express the human enzyme, with emphasis on studies of mouse kidney and lung. Mouse kidney and lung were studied using both frozen section analysis and paraffin sections following fixation in a variety of fixatives. Formalin fixation resulted in a loss of antigenicity, while fixation in zinc formalin or B5 fixative gave results similar to those from frozen sections. Immunoperoxidase studies using antibodies to MnSOD showed greater staining in transgenic kidney or lung than in identical tissues in normal mice when appropriate fixation was used. In contrast, equal immunostaining was obtained in kidney or lung from normal and transgenic mice when antibodies to catalase or copper zinc superoxide dismutase were utilized. Immunogold ultrastructural analysis of MnSOD localization for lung and kidney was also performed. As compared to normal mice, transgenic mice exhibited greater staining of the mitochondria of kidney interstitial fibroblasts and glomerular, endothelial, and smooth muscle cells. In the lungs of transgenic animals, all cells showed increased staining; smooth muscle cells demonstrated the most marked increase in immunolabelling. The results indicate that these transgenic mice overexpress MnSOD in their mitochondria, and that this occurs selectively in at least some mesenchymal tissues.This study was supported by the Medical Research Service of the Department of Veterans Affairs (TDO), by National Institutes of Health grants No. CA-41267 (LWO), No. HL-39585 and No. HL-44571 (Y-SH), and by the Department of Anesthesiology Research and Development Funds (DBC, HPC).     

Double labeling of SIgG+ cells harboring a lymphotropic herpesvirus by avidin-biotin complex immunoperoxidase and immunogold-silver staining techniques

By reversing the usual order of double-staining procedures, we obtained optimal conditions for simultaneous detection of guinea pig lymphotropic herpesvirus (GPHLV) and surface immunoglobulin G-positive (SIgG+) cells in paraffin-embedded tissue sections. Of the different fixatives tested, Bouin's solution gave the best preservation of morphology and cell surface IgG. Double immunolabeling was best achieved when avidin-biotin complex immunoperoxidase (ABC-IP) staining was performed first for detection of intracellular viral antigen, followed by immunogold-silver staining (IGSS) for localization of cell surface IgG.     

Detection of Cell Surface Antigens in Tissue Sections by Means of Pre-Embedding Immunogold Staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualise cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that it is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

Detection of cell surface antigens in tissue sections by means of pre-embedding immunogold staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualize cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

Use of pre-embedding ultrastructural immunocytochemistry in the localization of a secretory product and membrane proteins in cultured prolactin cells

A pre-embedding immunoperoxidase procedure performed directly on cultured cells in situ was used to localize several intracellular antigens at the electron-microscope level. With this procedure, we compared the effect of various fixatives, with or without saponin permeabilization, on the immunoreactivity of a secretory product (prolactin) and membrane proteins in cultured prolactin cells. Prolactin was detected within all compartments of its intracellular secretory pathway. Membrane antigens of the rough endoplasmic reticulum, the Golgi apparatus, and lysosomes were localized in distinct intracellular compartments. These immunocytochemical results are discussed in relationship to others in the literature that describe the localization of similar types of antigens. The technique, here described, which preserves ultrastructural detail and antigenicity, should be applicable for the localization of other intracellular antigens in cultured cells.     

Stability of avian leukosis virus type-specific antigen in the cell after fixation in periodate-lysine-paraformaldehyde

Stability of avian leukosis virus type-specific antigen in chicken embryo fibroblasts after fixation in periodate-lysine-paraformaldehyde was tested by the indirect immunoperoxidase method. Antigen that had been steeped in 0.015M Tris-HCl saline buffer solution containing 20% bovine serum and 30% glycerin was preserved at -20 degrees C, 4 degrees C and room temperature without loss of its antigenicity as long as 16 weeks. It was also stable against heat treatment at 56 degrees C, acid treatment and ethyl ether treatment.     

Complement activation induces the expression of decay-accelerating factor on human mesangial cells.

In the present study we evaluated the effect of complement activation by immune complexes (IC) on the expression of decay-accelerating factor (DAF) on human mesangial cells (MC). MC in culture were incubated with an Ag (DNP-Gelatin) that binds to fibronectin present in the MC matrix. Subsequently, MC were incubated with anti-DNP antibodies in the presence of human serum. By immunoperoxidase staining we showed that these incubations resulted in IC formation and deposition of human C3 and terminal complement components (C5b-9) on the mesangial matrix and on the surface of MC. By immunoperoxidase staining and by RIA we showed that IC formation and complement activation significantly increased DAF expression on the MC plasma membrane. The induction of DAF expression was a consequence of deposition of terminal complement components on the MC because, zymosan-activated serum and IC formation in the presence of C5- or C8-deficient serum failed to increase MC DAF expression. Furthermore, the observed increased DAF expression was the consequence of increased DAF synthesis by MC. Thus, both cycloheximide and actinomycin D blocked the increase on MC DAF observed after incubation with IC and serum. MC DAF had biophysical and functional characteristics similar to DAF in other cells. Thus, 1) MC DAF was resistant to trypsin but was removed from the MC membrane by pronase; 2) phosphatidylinositol-specific phospholipase C removed 48 +/- 4% of MC DAF indicating that MC DAF is anchored in the cell membrane by phosphatidylinositol groups; 3) DAF isolated from MC-inhibited complement-mediated hemolysis and demonstrated a molecular mass of 83 kDa. In conclusion, deposition of terminal complement components on human MC trigger new synthesis and membrane expression of DAF. Because DAF protects cells against complement-mediated lysis, we postulate that DAF may protect glomerular cells during IC and complement-mediated glomerulonephritis.     

Anti-double-stranded DNA antibodies and immunostimulatory plasmid DNA in combination mimic the endogenous IFN-alpha inducer in systemic lupus erythematosus

Patients with systemic lupus erythematosus (SLE) have increased blood levels of IFN-alpha, which correlate to disease activity. We previously identified an IFN-alpha-inducing factor (IIF) in the blood of SLE patients that activated the natural IFN-alpha-producing cells in cultures of normal PBMC. The SLE-IIF contained DNA and IgG, possibly as small immune complexes. In our study, we demonstrated that SLE-IIF correlated to the presence of anti-dsDNA Abs in patients and contained anti-dsDNA Abs as an essential component. Purified anti-DNA Abs or SLE-IgG caused only a weak IFN-alpha production in cultures of normal PBMC in the presence of costimulatory IFN-alpha2b. However, they converted the plasmid pcDNA3, which itself induced no IFN-alpha production in PBMC, into an efficient IFN-alpha inducer. A human monoclonal anti-ss/dsDNA Ab had the same effect. This IFN-alpha-inducing activity of the plasmid was abolished by methylation, suggesting that unmethylated CpG DNA motifs were important. Like IIF in SLE serum, the combination of SLE-IgG and pcDNA3 appeared to stimulate IFN-alpha production in natural IFN-alpha-producing cells, a unique cell population resembling immature dendritic cells. The IFN-alpha production was greatly enhanced by IFN-alpha2b and IFN-beta, and for SLE-IIF it was also enhanced by GM-CSF but inhibited by IL-10. We have therefore identified a new function of DNA-anti-DNA Ab complexes, IFN-alpha induction, that might be important in the pathogenesis of SLE.