MP26 in the bovine lens: a post-embedding immunocytochemical study

Gold immunolabeling of bovine lens tissue embedded in Lowicryl K4M, using a polyclonal antibody specific for a major component of lens fiber plasma membrane of 26 K molecular weight, shows that this constituent is absent from the epithelial cell plasma membrane and associated only with the junctional and non-junctional domains of the lens fiber plasma membrane.     

Optimal antigen localization in human tissues using aldehyde-fixed plastic-embedded sections

Although the utility of antigen labeling techniques in frozen tissues is well known, it is generally acknowledged that an improvement in morphologic preservation is desirable. Conventionally processed paraffin-embedded tissues are limited in the range of antigens that can be detected and newer plastic embedding techniques have been even more restricted. By using cold (4 degrees C) processing and limited fixation a wide range of antigens (including T and B markers) has been demonstrated in 2 mu plastic sections. The morphologic preservation and antigen localization are superior to other techniques. The combination of precise antigen localization and excellent morphologic preservation should expand the diagnostic and investigative uses of immunohistology.     

Macrophage Ia antigens. I. macrophage populations differ in their expression of Ia antigens

By indirect immunofluorescence and microcytotoxicity it was demonstrated that different populations of murine macrophages bear different amounts of Ia antigens on their membranes. At least three subpopulations could be distinguished: those that lack Ia antigens and predominate in peritoneal exudate; cells bearing I-A antigens that are the majority of splenic macrophages and a minor population in the peritoneum; and cells bearing I-C antigens that are a minor population in both spleen and peritoneum. Internal radioisotope labeling studies confirmed that the I region molecules are synthesized by the macrophages. It is suggested that these different macrophage subpopulations may play distinct roles in the immune response.     

Androgen receptor expression in human tissues: an immunohistochemical study.

The cellular localization of the human androgen receptor was visualized immunohistochemically using a mouse monoclonal antibody (MAb) F39.4, directed against a fragment of the N-terminal domain of the androgen receptor. The nuclear immunoreactivity of various human tissues with F39.4 was generally consistent with earlier biochemical and autoradiographic data. However, previously suggested androgen receptor expression in thyroid, pancreatic, gastrointestinal, and bladder tissues was not confirmed immunohistochemically. Stratified squamous epithelia of vagina and cervix showed selective immunostaining of the basal cell layer, whereas in the preputial epithelium the intensity of immunoreactivity decreased gradually with maturation. In contrast, glandular epithelia of the sweat glands, male accessory sex organs, and female breast showed nearly exclusive F39.4 staining of the inner cylindric layer. In the testis, Sertoli cells, peritubular myoid cells, and interstitial cells were immunoreactive with MAb F39.4. Expression of the androgen receptor by smooth muscle tissue was largely confined to the male reproductive organs. The specificity and sensitivity of this simple and rapidly performed immunohistochemical technique in the detection of the human androgen receptor at the cellular and subcellular level makes it worthwhile to study tissue androgen receptor expression by immunohistochemistry in physiological and pathological states.     

Brain Ia antigens have a bone marrow origin

Our results, using radiation-induced bone marrow chimeras, demonstrate that the Ia antigen found in the brains of such animals is produced by cells having precursors in the bone marrow. These cells are not immediately blood borne since no IgM is detected in these brains. This rules out the obvious possibility of B-lymphocyte contamination as the source of Ia in the brain cell preparations. It thus appears that the central nervous system, like many other nonlymphoid organs, has a source of Ia-positive cells that are derived from bone marrow precursors.     

Thomsen-Friedenreich-related carbohydrate antigens in normal adult human tissues: a systematic and comparative study

A broad variety of normal human tissues were examined for the expression of Thomsen-Friedenreich (TF)-related histo-blood group antigens. TF (Galβ1-3GalNAcα1-R), Tn (TF precursor, GalNAcα1-R), sialosyl-Tn (NeuAcα2-6GalNAcα1-R), considered to be useful in cancer diagnosis and immunotherapy, and sialosyl-TF, the cryptic form of TF. These antigens or, more correctly, glycotopcs, were determined by immunohistochemistry with at least two monoclonal antibodies (mAbs) each (except sialosyl-TF) as well as by lectin histochemistry. For a better dissection of sialosyl-TF and TF glycotopes, tissue sections were pretreated with galactose oxidase or the galactose oxidase-Schiff sequence. Staining with mAbs appeared to be more restricted than with the lectins used. Distribution patterns among normal epithelia were different for all four antigens. These antigens were also detected in some non-epithelial tissues. They can be classified in the following sequence according to the frequency of their occurrence in normal tissues: sialosyl-TF> >sialosyl-Tn>Tn>TF. Most of the positively staining sites for TF, Tn, and sialosyl-Tn are located in immunologically privileged areas. The complex results obtained with anti-TF mAbs (after treatment of the tissue sections with sialidase fromVibrio cholerae) and the lectins amaranthin and jacalin revealed a differential distribution of the subtypes of sialosyl-TF [NeuAcα2-3Galβ1-3GalNAcα1-R and Galβ1-3 (NeuAcα2-6)GalNAcα1-R] in normal human tissues. From our data it can be inferred that TF, Tn, and sialosyl-Tn are promising targets for a cancer vaccine.     

Human kallikrein 13 expression in normal tissues: an immunohistochemical study.

The human tissue kallikrein 13 gene (KLK13), encoding for hK13 protein, was recently cloned and characterized. Here we describe the immunohistochemical (IHC) localization of hK13 in normal human tissues and compare it with the expression of two other kallikreins, hK6 and hK10. We performed the streptavidin-biotin IHC method on 204 paraffin blocks from archival, current, and autopsy material prepared from almost every normal human tissue, using a polyclonal and a monoclonal hK13 antibody. The staining was cytoplasmic and both antibodies yielded similar results. The hK13 protein was revealed in a variety of tissues, mainly in glandular epithelia. Other epithelia that expressed hK13 included the urothelium, the spermatic epithelium, and the epithelium of the choroid plexus. hK13 was intensely immunoexpressed by some endocrine organs, such as the adenohypophysis, the thyroid gland, the parathyroid glands, the adrenal medulla, the Leydig cells of the testis, and the cells of the endocrine pancreas. Immunoreactivity was also observed in the primordial follicles, the corpus luteum, and sparse luteinized cells in the stroma of the ovary, the trophoblastic cells of the placenta, the Hassall's corpuscles of the thymus, and chondrocytes. Nerves and ganglia of the peripheral nervous system, and both neurons and glial cells in the central nervous system, were positive. In short, hK13 was expressed by many glandular epithelia, some endocrine organs, and some specialized epithelia and cells. Comparison of these data with hK6 and hK10 expression suggests that the three kallikreins have a similar IHC pattern in normal human tissues.     

Low-molecular-weight Ia antigens in normal mouse serum

Low-molecular-weight Ia antigens can be detected in mouse serum. A procedure for isolating these antigens from serum in high yield is described. The Ia antigen preparation was found to be rich in carbohydrate, low in protein, and strongly bound to Concanavalin A andLotus lectins. Furthermore, the Ia antigenicity was destroyed by periodate oxidation and neuraminidase treatment, but was unaffected by pronase. These observations strongly suggest that the Ia antigens in serum are oligosaccharide in nature. Such a conclusion implies that at least some of the genes in theI region of theH-2 gene complex code for glycosyl transferase enzymes.     

Enzyme histochemical demonstration of alkaline phosphatase activity in plastic-embedded tissues using a Gomori-based cerium-DAB technique

We describe a new method for light microscopic demonstration of alkaline phosphatase (ALP) activity in plastic-embedded sections. Rat tissues were fixed in acetone (-20 degrees C), infiltrated in glycol methacrylate (GMA), and embedded at 0 degrees C. Sections were cut at 1 and 2 microns, dried at room temperature, and incubated in the conventional Gomori medium. Cerium chloride was used to convert calcium phosphate into cerium phosphate, which was subsequently converted into cerium perhydroxide. The slight yellow precipitate of cerium perhydroxide was amplified using 3,3'-diaminobenzidine tetrahydrochloride (DAB). For comparison, tissue sections were processed according to the calcium-cobalt method. The method described combines exact localization of ALP activity with optimal preservation of tissue morphology.     

Protein phosphatase 2Calpha expression in normal human tissues: an immunohistochemical study

The recent development of fluorescent proteins has rapidly and radically altered the way cell biology is performed by allowing simple, non-invasive imaging of cellular processes in real time. The special properties of the nervous system, such as synaptic morphology, axonal/dendritic maturation, and neuronal migration are especially amenable to investigation using fluorescent proteins. This review focuses on the various genetic and viral vectors used to express fluorescent proteins in vivo and in slice culture, and the strengths and limitations associated with them.     

Blood groups antigens distribution in pancreatic cancer. An immunohistochemical study

Distribution and immunolocalization of blood groups antigens ABO were investigated in pancreatic cancer with the use of immunoperoxidase method. In contrast to earlier investigations no correlation was found between histological differentiation of pancreatic cancers and maintenance of the blood groups antigens in tumor cells.     

Characterization of Ia antigens in mouse serum.

Sera obtained from normal B10.BR mice were shown to inhibit selectively a specific anti-Ia alloantiserum.Partial purification of the Ia antigenic activity was accomplished by isolation of the high density lipoproteins from these sera by fractional precipitation with sodium phosphotungstate and MgCL2. Both H-2.23 and Iak antigens present in this high density lipoprotein fraction were completely adsorbed by rabbit anit-rat beta2-microglobulin immunoadsorbents, whereas specific anti-H-2.23 immunoadsorbents removed only the H-2 activity. These data deomnstrate that Ia antigens, like H-2 antigens in the sera of B10.BR mice are associated with high density lipoproteins and further suggest that both H-2 and Ia antigens are associated with a beta2-microglobulin-like molecule.     

Ia antigens: markers of specific T suppressors immune to H-2 complex antigens

Two antisera to Ia antigens, products of the H-2 complex I-Cd and I-JkEk subregions, respectively, have been obtained by immunisation of the F1 hybrids of recombinant strains of mice. These antisera are shown to display the 50 per cent cytotoxic effect in vitro in the presence of complement upon lymphocyte populations immune to the H-2 complex antigens and enriched for specific suppressor T cells (SSC) by fractionation on the monolayer of target cells. The specificity of anti-Ia cytotoxins is shown by the cross antibody absorption with T- and B-cells of mice originated from the recombinant H-2 haplotypes and bearing either particular I-Cd, I-Jk and I-Ek antigens, or their combinations. Anti-I-Cd cytotoxins are found to react with both B and T cells at a different rate, and the anti-I-JkEk serum contains two antibody types directed to I-Ek and I-Jk products, respectively, the latter being able to react preferently with T cells. Although both antisera do inactivate the in vitro SSC function in the presence of complement at a similar degree, the inactivating action of the anti-I-Cd serum, but not that of the anti-I-JkEk serum, occurs without complement. SSC are established to bear both Ia-antigens, I-J and I-C on the same cell, as demonstrated by the cross antibody absorption and variation of the H-2 origin of SSC. These two markers are suggested to function differently in the SSC immune to the H-2 antigens and the I-C antigen expression on the SSC surface is presumed to be required for their interaction with the inhibited responder T cells proliferating in MLC.     

The expression of Ia antigens on immunocompetent cells in the guinea pig. II. Ia antigens on macrophages.

Only 15 to 25% of purified oil-induced guinea pig macrophages could be lysed by treatment with anti-Ia serum and C. Those cells remaining alive after treatment were not damaged and were metabolically active since they readily phagocytized latex beads. However, the "Ia-negative" macrophages were markedly deficient in their ability to present protein antigens to immune T lymphocytes and to function as stimulator cells when mixed with allogeneic T cells in the mixed leukocyte reaction. It thus appears that Ia antigens are expressed on a subpopulation of macrophages and that this subpopulation plays a critical role in the activation of T cell proliferation to soluble protein antigens and to alloantigens.     

Ag-B-linked analogue of Ia antigens in the rat.

The reactions of Lewis rat lymphocyte membrane antigens with two alloantisera, BN anti-Lewis and BN anti-Fischer have been studied. Three lines of evidence indicated that these antisera reacted with cell surface antigens homologous to Ia antigens of the mouse. 1) After absorption with Lewis platelets, the antisera killed only 40 to 50% of Lewis spleen cells. The majority of such cells were shown to be Ig-positive B cells by the examination of reaction patterns on lymphocytes after separation on nylon wool into T cell- and B cell-enriched subpopulations. 2) SDS-PAGE analysis of solubilized Lewis spleen cell antigens precipitated with these antisera revealed that the platelet-absorbed antisera reacted with molecules comparable in size to mouse Ia antigens (mw approximately equals 35,000 and 28,000). The unabsorbed sera reacted with these molecules and with additional molecules corresponding in size to mouse K and D antigens (m.w. = 45,000). 3) Neither of these antisera killed significant numbers of spleen cells from the partially congenic strain F.BN (seventh backcross homozygotes), a Fischer rat to which the Ag-B.3 allele is being transferred by repetitive backcrossing, indicating that the genes coding for these Ia-like antigens in the rat are linked to the Ag-B locus.     

Distribution of vitamin B12 R binder in normal human tissues: an immunohistochemical study

We studied the distribution of vitamin B12 R binder in various normal human tissues by use of an immunoperoxidase technique. Positive staining for R binder was observed in almost all glandular epithelia of digestive system, bronchial glands, renal proximal tubules, prostate, uterus, Fallopian tube, mammary gland, and sweat glands. The distribution of R binder was similar to that of lactoferrin and secretory component. These findings support the hypothesis that R binder plays a role in the local defense mechanism.     

Choice of fixatives for the immunohistochemical study of antigens using immunoadsorption

The effect of fixatives on the rat bone marrow antigens was studied by a method combining immunoadsorption and immunodiffusion. This method allows to estimate rapidly and reliably the effect of fixation on immunochemical properties of the antigen under study, using polyvalence antisera. Acetone and ethanol proved to be the best fixatives for the rat bone marrow antigens since they preserved their immunochemical properties. Aldehydes (formaldehyde and glutaraldehyde) "inactivated" the bone marrow antigens.     

Presence of chromogranin A, B and C in bovine endocrine and nervous tissues: a comparative immunohistochemical study

Summary Antisera against chromogranin A, B and C were used to study the distribution of these acidic proteins in bovine endocrine and nervous tissues. The three chromogranins occur together in several endocrine organs (adrenal medulla, anterior pituitary, endocrine pancreas) and in sympathetic ganglion cells. In the posterior pituitary, only chromogranin C and in the intermediate lobe only A and C are found. The parathyroid gland contains only A, and enterochromaffin cells are immunoreactive for A and B. Cells of the thyroid gland and some cells of the anterior pituitary apparently do not contain any chromogranins. It is concluded that the three chromogranins are not always stored together and that they are not present in all endocrine cells. This distinct localization of the chromogranins indicates some special, although still undiscovered, function for these proteins.     

A role for Ia antigens in thymocyte binding by macrophages

To investigate the membrane structures involved in cellular interactions between thymocytes and macrophages, the relative ability of different murine macrophage populations to spontaneously bind thymocytes was compared. Macrophages derived from the spleen or thymus bound three to four times the number of thymocytes than macrophages from peripheral blood, peritoneum, or bone marrow. This reflects differences both in the number of macrophages binding thymocytes and in the number of thymocytes bound per macrophage. The extent of binding seems to positively correlate with the number of Ia-positive macrophages contained in these populations, as based on previously published values. This was confirmed by showing that elimination of splenic Ia-positive macrophages with anti-Ia and complement treatment dramatically reduced thymocyte binding. In addition, mouse peritoneal washout macrophages incubated for several days with supernatant fluid from concanavalin A-stimulated spleen cells, which induce Ia-antigen expression, exhibited a marked increase in the number of macrophages that bound thymocytes and the number of thymocytes bound per macrophage. To determine if Ia antigens were directly involved in binding, spleen, thymus, or Ia-induced peritoneal macrophages were treated with a monoclonal anti-Ia antibody prior to the addition of thymocytes. Treatment with anti-Ia reduced binding by around 50%, whereas treatment with anti-H-2D antibody had no effect. Monoclonal anti-I-A and anti-I-E antibody treatments of macrophages both inhibited thymocyte binding to similar extents, and treatment of macrophages with both reagents together reduced thymocyte binding by 80%. These results indicate that thymocyte binding is in part dependent on macrophage Ia expression.