HLA antigens on human hemopoietic progenitors during embryonic-fetal life: expression and in vitro modulation

We have analyzed the time course of the expression of HLA antigens on hemopoietic progenitors (BFU-E, CFU-E and CFU-GM) from human embryonic-fetal livers (FL). HLA ABC and Ia-like antigens are never detected on progenitor cells at 5-week post-conception. Their expression initiates at 6-week and progressively increases thereafter, up to near-adult levels at 9-week. The time course of HLA antigen expression on erythroid precursors, derived from FL at corresponding gestational ages, parallels that observed on hemopoietic progenitors. Incubation of embryonic progenitors or precursors with medium conditioned by PHA-stimulated adult peripheral blood mononucleated cells induces a marked increase of the expression of both HLA-ABC and Ia-like antigens. Conversely, incubation with recombinant gamma-interferon causes a marked increase of HLA-ABC, but not Ia-like antigen expression, thus in contrast with results observed on adult monocytes.     

Bromodeoxyuridine and lododeoxyuridine Double Immunostaining for Epoxy Resin Sections

To establish bromodeoxyuridine (BrdUrd)/iododeoxyuridine (IdUrd) double immunostaining for thick sections of epoxy resin-embedded tissues, young hamsters received intra-peritoneal injections of IdUrd and BrdUrd 3 hr and 1 hr before sacrifice, respectively. The intestines were fixed with phosphate-buffered 4% paraformaldehyde and embedded in an Epon-Araldite mixture. The epoxy resin was removed completely by a sodium methoxide/benzene/methanol solution. This epoxy resin removal method was effective for BrdUrd/IdUrd immunostaining using a mono-specific antibody for BrdUrd (Br-3) and a bi-specific antibody for BrdUrd and IdUrd (IU-4), followed by the ABC complex method. Epoxy sections stained with these antibodies showed clear localization of nuclei incorporating the two thymidine analogues with precise morphology of labeled cells. Furthermore, ultrastructural observation of thin sections adjacent to thick sections immunostained for BrdUrd/IdUrd confirmed the cell type and ultrastructural features of cells labeled with these thymidine analogues.     

Combination of the peroxidase anti-peroxidase (PAP)-and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining

Summary A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step — Incubation of the tissue sections with the monoclonal primary antibodies; Second step — biotinylated anti-rat or anti-mouse IgG; Third step — monoclonal PAP complex; Fourth step — ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step — the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4-and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells. Stronger staining intensity was obtained for desmin-like immunoreactivity (LI) (within myofibroblasts of the lamina propria and muscle cells of the blood vessels)-and vimentin-LI (within Sertoli cells, myofibroblasts of the lamina propria and fibroblasts of the interstitium) of the human testis. The full combination (6 step-reaction) permits the detection of smallest quantities of an antigen in sections of different tissues using highly diluted primary antibodies. The technique represents a further alternative among the immunocytochemical amplification methods.     

CD56(NCAM) antigen in glandular epithelium of human thyroid: light microscopic and ultrastructural study

CD56 antigen, an isoform of the neural cell adhesion molecule (NCAM) was previously found by us in human thyroid by APAAP immunohistochemistry in light microscopy on frozen tissue sections. In the current study, it was attempted to trace the antigen in question using another light microscopic immunohistochemical procedure and to validate the results at the ultrastructural level. For light microscopy, cryostat sections of 12 surgical samples of human thyroid were subjected to ABC (preformed avidin-biotin-peroxidase complex) method. For immunoelectron microscopy, immunoperoxidase reaction was carried out on prefixed, small thyroid tissue blocks. Following preliminary inspection of semithin sections, ultrathin sections were examined in the transmission electron microscope. ABC reaction revealed distinct specific CD56 staining of thyrocyte cell membranes. The staining was weak or absent in thyroid papillary carcinoma cells. The results were confirmed in semithin sections by indirect immunoperoxidase. The latter reaction in ultrathin sections at the ultrastructural level has shown that specific reaction product was confined to free and lateral surfaces of thyroid follicular cells. Endothelial cell membranes of thyroid capillary vessels were totally devoid of the reaction product. The reaction was weakly positive in thyroid follicular and papilllary carcinomas but absent from medullary carcinoma.     

Masking of sperm maturation antigen by sialic acid in the epididymis of the mouse. An immunohistochemical study

Sperm antigen expression during epididymal transit was examined in 4- to 16-week-old intact and castrated ICR mice, using the avidin-biotin complex (ABC) immunohistochemical method with monoclonal antibody T21 against a flagellar surface antigen. On untreated sections, the antigen was first expressed weakly on sperm in the proximal part of the corpus epididymis, and intraluminal components were stained in 4-week-old mice. Epididymal epithelial cells and their stereocilia, and cells in other reproductive organs were not stained. In contrast, on sections treated with neuraminidase, (1) the initial site of antigen appearance is a more proximal position in treated than in untreated sections, (2) stereocilia stained strongly, (3) the staining intensity of sperm and intraluminal components increased, and (4) some clear cells in the epithelium from the distal position of the caput to the corpus epididymis were stained. These results indicate that the antigen is produced by clear cells of the epididymal epithelium, that the antigenic determinant is masked initially by sialic acid residues, and that expression of the antigenic determinant on the sperm surface during epididymal maturation apparently involves desialylation.     

Serological cross-reactivity of murine A.TH anti-A.TL antibody (anti-l-Ek antibody) with La-like molecules of human antigen-presenting cells

The purified protein derivative (PPD)-specific proliferative responses of T cells from human peripheral blood are shown to be dependent on antigen-presenting cells (APC) which bear HLA-DR antigen detected by the monoclonal anti-HLA-DR antibody. The serological cross-reactivity of murine A.TH anti-A.TL antibody was observed in human APC. By absorption experiments using H-2 congenic mice, the serological cross-reactivity of A.TH anti-A.TL antibody with human APC is mapped in the I-E subregion. Thus, anti-I-Ek antibody reacts with the Ia-like molecule(s) on human APC. Murine allo-anti-I-Ek antibody does not always react with determinants of Ia-like molecule(s) on human APC, since this antibody did not eliminate PPD-specific proliferative responses in one particular case. Thus, anti-I-Ek antibody seems to react some type of the polymorphic determinants but not of the shared determinants of human Ia-like molecule(s) on APC. The relationship between the cross-reactive molecule detected by murine allo-anti-I-Ek antibody and the HLA-DR antigen remains to be analyzed.     

Leu-7(HNK-1)在人胃肠内分泌细胞中的表达

Leu-7(HNK-1)是一种分子量为110KD的糖蛋白,它是人类NK和K细胞特异性的表面抗原。Leu-7单克隆抗体可特异性识别NK和K细胞表面抗原。本文应用ABC免疫组织化学染色技术研究了Leu-7在人胃肠道的定位和分布。结果发现,Leu-7免疫反应(Leu-7-IR)细胞主要分布于胃腺和肠腺的底部。偶见于肠绒毛上皮中,呈典型内分泌细胞的形态特征。相邻切片法免疫双标记证明多数胆囊收缩素(CCK-8),生长抑素(SS),胃泌素(G34)或5-羟色胺(5-HT)免疫反应阳性细胞呈Leu-7阳性。我们的发现支持了在神经,内分泌和免疫系统间存在相同或相似抗原决定簇的假说。     

Combination of the peroxidase anti-peroxidase (PAP)- and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining.

A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction: First step--Incubation of the tissue sections with the monoclonal primary antibodies; Second step--biotinylated anti-rat or anti-mouse IgG; Third step--monoclonal PAP complex; Fourth step--ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step--the ABC complex. Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4- and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Masking of sperm maturation antigen by sialic acid in the epididymis of the mouse

Summary Sperm antigen expression during epididymal transit was examined in 4- to 16-week-old intact and castrated ICR mice, using the avidin-biotin complex (ABC) immunohistochemical method with monoclonal antibody T21 against a flagellar surface antigen. On untreated sections, the antigen was first expressed weakly on sperm in the proximal part of the corpus epididymis, and intraluminal components were stained in 4-week-old mice. Epididymal epithelial cells and their stereocilia, and cells in other reproductive organs were not stained. In contrast, on sections treated with neuramainidase, (1) the initial site of antigen appearance is a more proximal position in treated than in untreated sections, (2) stereocilia stained strongly, (3) the staining intensity of sperm and intraluminal components increased, and (4) some clear cells in the epithelium from the distal position of the caput to the corpus epididymis were stained. These results indicate that the antigen is produced by clear cells of the epididymal epithelium, that the antigenic determinant is masked initially by sialic acid residues, and that expression of the antigenic determinant on the sperm surface during epididymal maturation apparently involves desialylation.     

The unlabeled antibody method: comparison of peroxidase-antiperoxidase with avidin-biotin complex by a new method of quantification

Upon plotting of areas against optical densities in immunocytochemically stained tissue sections, hyperbolic curves were obtained which could be reduced to two straight lines, one representing variations in stained structures, and the other variations in background. The slopes of the stained structure lines reflected staining intensity independently of total area of stained structure in a section. The ratio of slopes of the stained structure and background lines reflected immunocytochemical sensitivity. A comparison of the peroxidase-antiperoxidase (PAP) method with the avidin-biotin complex (ABC) method showed that at usual antibody dilutions the PAP method was much more sensitive than the ABC method, while at impractically high antibody dilutions it was moderately more sensitive. Once sufficient dilutions of antibodies were reached, staining intensities dropped sharply with the PAP method. On the other hand, the dilution curves were flat with the ABC method. The ABC method consequently appeared unsuitable for estimating variations in concentration of antigen or for distinguishing high or low concentrations of antigen. The ABC method provided a stain for myelin even in the absence of any antibodies.     

Monoclonal antibodies to HLA-A,B, and Ia-like antigens inhibit colony formation by human myeloid progenitor cells

Hybridomas derived from the fusion of murine myeloma cells with splenocytes from mice immunized with human cultured lymphoid cells secreted monoclonal antibodies to human cell surface antigens. Serologic and immunochemical assays showed that 4 monoclonal antibodies (Ab Q2/47, Q2/61, Q2/70, Q2/80) recognize framework determinants of Ia-like antigens and 1 monoclonal antibody (Ab Q1/28) reacts with determinants expressed on the heavy chain of HLA-A,B antigens. Both anti-HLA-A,B and anti-Ia-like antigen monoclonal antibodies caused complement-dependent inhibition of granulocyte-macrophage colony formation by human bone marrow grown in soft agar. Mixing experiments excluded the possibility of an indirect effect on progenitor cells by lysis of auxiliary cells. These results indicate that human myeloid progenitor cells express HLA-A,B and Ia-like antigens.     

免疫酶双重染色中抗体洗脱的进一步研究

本实验进一步检查了三种常用的洗脱方法从切片上除去免疫酶组织化学染色后的抗体的效果。结果表明,PAP法染色后,氧化法的抗体洗脱完全,效果可靠;酸洗法和酸洗/二甲基甲酰胺法的效果视不同抗体而不同,二甲基甲酰胺单用或用于酸洗后似无效。所用方法均不能从ABC法染色的垂体组织切片上完全除去抗体复合物。因之,将ABC法用于第一抗体来源于同一动物种的双重染色中,来显示第一种抗原时,要特别注意排除假性双标记。     

Optimizing collagen antigen unmasking in paraffin-embedded tissues

In order to optimize collagen antigen unmasking in paraffin-embedded tissue sections, the effects of various fixatives and duration of fixation in relation to enzyme pretreatment and microwave irradiation for collagen antigen unmasking were studied. A streptavidin--biotin-- peroxidase complex method was used for the immunolocalization of type III and IV collagen antigens. Fixatives and fixation time had significant adverse effects on the immunoreactivity of the antigens. Enzyme pretreatment was found to be superior to microwave irradiation for collagen antigen unmasking. Fixation with paraformaldehyde required shorter enzyme pretreatment and yielded a more enhanced reaction than treatment with formalin and Bouin's fluid. The optimum conditions for type III and IV collagen unmasking were found to be fixation with 4% paraformaldehyde in 0.01 m phosphate-buffered saline, pH 7.4, for up to 3 weeks followed by enzyme pretreatment with 1 mg ml−1 pepsin in 0.01 n hydrochloric acid, pH 2.0, for 30 min (human tissues) or 60 min (rat tissues) at 37°C. It is concluded that collagen antigen unmasking by enzyme pretreatment in tissue sections fixed for a long period of time can be successful if appropriate enzyme(s) and incubation time(s) are employed with regard to the antigen under study and fixative and fixation time used for tissue preparation     

Optimizing collagen antigen unmasking in paraffin-embedded tissues

In order to optimize collagen antigen unmasking in paraffin-embedded tissue sections, the effects of various fixatives and duration of fixation in relation to enzyme pretreatment and microwave irradiation for collagen antigen unmasking were studied. A streptavidin--biotin-- peroxidase complex method was used for the immunolocalization of type III and IV collagen antigens. Fixatives and fixation time had significant adverse effects on the immunoreactivity of the antigens. Enzyme pretreatment was found to be superior to microwave irradiation for collagen antigen unmasking. Fixation with paraformaldehyde required shorter enzyme pretreatment and yielded a more enhanced reaction than treatment with formalin and Bouin's fluid. The optimum conditions for type III and IV collagen unmasking were found to be fixation with 4% paraformaldehyde in 0.01 m phosphate-buffered saline, pH 7.4, for up to 3 weeks followed by enzyme pretreatment with 1 mg ml−1 pepsin in 0.01 n hydrochloric acid, pH 2.0, for 30 min (human tissues) or 60 min (rat tissues) at 37°C. It is concluded that collagen antigen unmasking by enzyme pretreatment in tissue sections fixed for a long period of time can be successful if appropriate enzyme(s) and incubation time(s) are employed with regard to the antigen under study and fixative and fixation time used for tissue preparation     

Tissue distribution of human NK cells studied with anti-Leu-7 monoclonal antibody

The distribution and numbers of human natural killer (NK) cells in different lymphoid tissues and several tumors were determined with the use of monoclonal antibody to the Leu-7 antigen and immunohistologic methods. Anti-Leu-7 reacts with large granular lymphocytes containing most of NK activity in the peripheral blood. The Leu-7+ cells were found mainly in the germinal centers of secondary follicles in lymph nodes, spleens, and tonsils. The mean number of Leu-7+ cells in 40 germinal centers was 223 +/- 103 SD. Only rare cells were found in the thymus. In the follicles studied in serial sections, the distribution of the Leu-7+ cells was distinctly different from that of T cells, B cells and Ia+ cells. The Leu-7+ cells did not react with OKT 6 antibody specific for immature (control) thymocytes and Langerhans' cells. By double staining techniques, two populations of Leu-7+ cells were identified in lymphoid tissues: those that did and did not express the Ia-like antigen. The numbers and localization of the Leu-7+ cells varied in different tumors, but often the Leu-7+ cells surrounded the neoplastic nodules or were seen in contact with individual cancer cells. Anti-Leu-7 may be a useful reagent for monitoring the presence and localization of NK cells in normal and malignant human tissues.     

Immunohistochemical demonstration of surface antigen of human lymphocytes with monoclonal antibody in acetone-fixed paraffin-embedded sections

Although the majority of reported studies have used fresh-frozen sections in detecting surface antigen of lymphocytes in tissue via monoclonal antibody, detailed histological figures can not be obtained by this method. Nor can the antigenicity be preserved for any length of time. A new method for detecting the surface antigen of lymphocytes using fixed and embedded material is presented. Human spleens were fixed in cold acetone, embedded in low melting point paraffin wax, and the thin sections treated with hyaluronidase. Anti-T lymphocyte monoclonal antibody (anti-Leu-1, anti-Leu-2, anti-Leu-3) and anti-HLA-DR were applied on these sections, and the antigen was detected by the ABC (avidin-biotin-peroxidase complex) method. The results were then compared with those of fresh-frozen sections. There was no great difference in detecting T and B cells or their subsets, but the histological figures were substantially better preserved in sections prepared by the present method. Furthermore, the antigenicity was retained in the materials fixed and embedded for more than two years.     

A comparison of three immunoperoxidase techniques for antigen detection in colorectal carcinoma tissues

We compared the streptavidin-peroxidase conjugate (SP) method of immunoperoxidase histochemistry to the unlabeled antibody (PAP) and avidin-biotin-peroxidase complex (ABC) techniques in human colorectal carcinoma tissues stained with a monoclonal antibody for expression of carcinoembryonic antigen. Compared to the ABC and PAP method, the SP method produced stronger staining intensity and very low background staining. This was true when other antibody isotypes, other antibody species, other organs, and another tumor-associated antigen were used. Moreover, the SP procedure time could be reduced to one third that of the ABC or PAP methods without compromising accuracy, and the SP reagent is stable for several months. The chemical nature of the streptavidin molecule accounts, in large part, for the advantages of the SP method.     

Fixation of cryo-sections under HIV-1 inactivating conditions: integrity of antigen binding sites and cell surface antigens

Summary Cryostat-sections of biopsies from HIV-infected patients or HIV/SIV-infected experimental animals pose a biohazard risk to laboratory workers. The objective of this study was to select a procedure that appropriately fixes cryo-sections and reduces the risk of HIV-1 infectivity. This inactivation procedure should preserve antigen binding capacity of host-produced antibodies and the antigenic structure of epitopes present in these tissues, while retaining sufficient morphologic detail. We tested the effect of seven different established fixation-inactivation procedures for HIV-1 on the detection of specific antibodies and membrane markers, compared to acetone fixation as a reference. Frozen sections of spleens from mice immunized with trinitrophenyl (TNP)-Ficoll were incubated with TNP-alkaline phosphatase to detect specific antibody-forming cells and follicular immune complexes containing TNP-specific antibodies. In addition, sections were stained with monoclonal antibodies directed against IgM (187-1), T-cells (anti Thy-1), and marginal metallophilic macrophages (MOMA-1). Five procedures proved useful as they gave results similar to regular acetone fixation. In contrast, two procedures with a methanol-containing fixative obscured both antigen binding sites and membrane antigens. Subsequently, these five selected procedures were tested on glass slide preparations of HIV-1 infected cell lines, expressing HIV-1 determinants defined by monoclonal antibodies. Finally, the procedures were tested on sections of an HIV-1 infected human lymph node. for detection of HIV-specific B-cells. We show that fixation-inactivation in 0.37% (v/v) formaldehyde in PBS for 10 min at room temperature and 0.5% paraformaldehyde (w/v) in PBS for 10 min at room temperature are the methods of choice, combining preservation of antigen binding sites (Fab), membrane antigens, and HIV-1 determinants with good tissue morphology.Abbreviations AFC antibody forming cell - AP alkaline phosphatase - MAb monoclonal antibody - HIV-1 human immunodeficiency virus type 1 - HRP horseradish peroxidase - TNP trinitrophenyl     

Conditions for the immunohistochemical demonstration of complement factor C3 in formaldehyde-fixed and paraffin-embedded renal tissues

Paraffin sections of formaldehyde-fixed renal biopsies were labeled for complement C3 by a polyclonal rabbit antibody to human complement C3, by the peroxidase-antiperoxidase complex (PAP) and the avidin-biotin peroxidase complex (ABC) techniques, respectively. All tissues had C3 deposits according to direct immunofluorescence on fresh frozen sections. Staining for muramidase was introduced as an intrinsic control for the degree of tissue proteolysis after the necessary trypsin digestion prior to the immunoenzyme labeling. The results indicated that even minute deposits of C3 could be detected in paraffin sections by the ABC method, which was more sensitive than the PAP technique; the ABC method allowed a maximal dilution of 1:2,400 of the primary antibody as compared to 1:800 for the PAP technique.     

Evaluation of immunohistochemical staining of human duodenal endocrine cells after microwave antigen retrieval

Summary The effect of microwave antigen retrieval on the immunostaining of human duodenal endocrine cells in formaldehyde-fixed, paraffin-embedded material was investigated. The sections were immunostained by the avidin-biot in complex (ABC) and immunogold-silver autometallography (IGSS) methods with and without prior microwave treatment. Dilutions of up to 1:30 000 of the following antisera/antibodies were used: anti-chromogranin A, anti-chromogranin AB, anti-secretin, anti-gastrin, anti-gastric inhibitory polypeptide, anti-somatostatin and anti-serotonin. The detection threshold for all the antibodies was lower after antigen retrieval, and the primary antibody could be used in higher dilutions. The dilutions varied for different antibodies and were between two and ten times the optimal dilution without antigen retrieval. At extremely high dilutions of, or without, the primary antibody, non-specific staining of some lymphocytes and the mucus of some goblet cells was observed when the avidin method was applied, but not with the immunogold technique. This phenomenon was not observed when optimal dilution or a lower dilution was used. This seems to have been caused by the binding of the avidin-biotin complex to epitopes in these structures unmasked by microwave treatment when competition with specific binding sites was absent. This revised version was published online in November 2006 with corrections to the Cover Date.