Double immunocytochemical staining in the study of antibody-producing cells in vivo. Detection of specific antibody-producing cells in the spleen and simultaneous determination whether or not they produce immunoglobulin G antibodies

Rabbits were primed intravenously with human serum albumin (HSA) and boosted with the same antigen 2 months later. Cells producing specific antibodies against HSA could be detected in vivo and it could be determined whether or not they belonged to the immunoglobulin (Ig) G class using a combined peroxidase (HRP) and alkaline phosphatase (AP) immunocytochemical technique. HRP-HSA conjugate was used for detection of anti-HSA-producing cells and AP-sheep anti-rabbit IgG (SRIgG) was used to determine the IgG class of the antibodies produced by these cells in the same spleen section. After performing both HRP and AP cytochemistry, cells with a red-stained cytoplasm represent anti-HSA-producing cells not stained for their antibody class and cells with a blue-stained cytoplasm represent cells producing IgG antibodies not directed against HSA. Cells with a double-stained cytoplasm represent cells producing anti-HSA antibodies belonging to the IgG class. We also attempted to determine whether or not part of the anti-HSA-producing cells belonged to the IgM class using AP-sheep anti-rabbit IgM (SRIgM). In this case no double-stained cells were detected, indicating that the affinity of intracellular IgM-anti-HSA antibodies is too low to allow detection using the present technique.     

Double immunocytochemical staining in the study of antibody-producing cells in vivo. Combined detection of antigen specificity (anti-TNP) and (sub)class of intracellular antibodies

Mice and rabbits were immunized with trinitrophenyl (TNP)-conjugated keyhole limpet hemocyanin (KLH). Cells producing specific antibodies against the hapten TNP were detected in vivo in spleen and lymph nodes using a TNP--alkaline phosphatase (AP) conjugate. Using horseradish peroxidase (HRP)-conjugated anti-mouse (sub)class (IgG2A, IgG2B, IgM) antibodies and anti-rabbit class (IgG, IgM) antibodies and a double immunocytochemical staining technique for simultaneous demonstration of the enzymes AP and HRP, we were able to determine both the antigen specificity (anti-TNP) and the (sub)class of intracellular antibodies produced by individual antibody-forming cells in vivo.     

Double immunocytochemical staining for in vivo detection of epitope specificity and isotype of antibody-forming cells against synthetic peptides homologous to human immunodeficiency virus-1.

Many infections evoke a strong humoral immune response. Some (e.g., HIV-1, EBV, CMV) also lead to disorders of the B-cell system. Data concerning cell dysfunction are largely derived from in vitro studies, which necessarily exclude all microenvironmental influences. The aim of this study was to develop a tool for the investigation of epitope specific humoral immune responses in vivo. Mice were immunized with one of two synthetic peptides, both 21 amino acids long and homologous to regions of the HIV-1 gp160. Cryostat sections of spleen and lymph nodes were incubated with the corresponding peptide coupled to alkaline phosphatase and simultaneously incubated with peroxidase-conjugated rabbit antisera specific for mouse immunoglobulin isotypes. We were able to show simultaneous detection of epitope specificity, isotype, and localization of antibody-forming cells and immune complexes in tissue sections. It should prove useful for in vivo investigation of the development of specific (e.g., anti-HIV-1) humoral immune response, the determination of B-cell specificity in lymph node infiltrates, and the role of immune complexes in lymph node pathology.     

Double immunocytochemical staining for the in situ study of allotype distribution during an anti-trinitrophenyl immune response in chimeric rabbits

After incubation of tissue sections with anti-allotype-enzyme conjugates, the localization of immunoglobulin-allotype-bearing cells in the lymphoid tissues of conventional and chimeric rabbits could be established. The use of anti-allotype sera bearing distinct enzyme labels allowed simultaneous recognition of B cells producing immunoglobulin of one or the other parental types in heterozygous rabbits, or of B cells from the donor and recipient in chimeras. After immunization of chimeric rabbits with trinitrophenyl-keyhole limpet hemocyanin, anti-trinitrophenyl antibody-forming cells could be demonstrated through the use of a trinitrophenyl-alkaline phosphatase conjugate. Simultaneous incubation of sections with this reagent and with horseradish peroxidase coupled to (donor or recipient) anti-allotype sera made possible the determination of the origin (donor or recipient) of the antibody-forming cells. In agreement with the results of plaque assays and analyses of serum antibodies, all the anti-TNP producing cells were of donor origin when the chimeras had been created through injection of spleen or lymph node cells from trinitrophenyl primed donors. With this study we introduce a simple, direct method for the simultaneous identification of cells that produce antibody of a given allotype and a given specificity, applicable to appropriate studies in heterozygous or chimeric rabbits. The procedure has various advantages over previously reported methods.     

Studies with anti-bromodeoxyuridine antibodies: II. Simultaneous immunocytochemical detection of antigen expression and DNA synthesis by in vivo labeling of mouse intestinal mucosa

We developed a rapid and convenient immunocytochemical method for simultaneous detection of antigen expression and S-phase cells by means of anti-bromodeoxyuridine (BrdUrd) antibodies. Immunocytochemical detection of BrdUrd after in vivo administration in mice was compared with autoradiography using [3H]-BrdUrd. Both the sensitivity and specificity of the technique were high. For the dual peroxidase staining technique, DAB color modification by cobalt ions was used. We showed that antigen localization was not affected by the BrdUrd staining protocol. The technique we describe here can be performed on frozen or paraffin-embedded tissue and on cytocentrifuge preparations for analysis of the cytokinetics of phenotypically defined cells in heterogeneous populations.     

Processing the nonstructural polyproteins of Sindbis virus: study of the kinetics in vivo by using monospecific antibodies.

Plasmids were constructed which contained a large portion of each of the four nonstructural genes of Sindbis virus fused to the N-terminal two-thirds of the trpE gene of Escherichia coli. The large quantity of fusion protein induced from cells containing these plasmids was subsequently used as an antigen to generate polyclonal antisera in rabbits. Each antiserum was specific for the corresponding nonstructural protein and allowed ready identification of each nonstructural protein and of precursors containing the sequences of two or more nonstructural proteins. These antisera were used to determine the stability of the mature nonstructural proteins and to examine the kinetics of processing of the nonstructural proteins from their respective precursors in vivo. Pulse-chase experiments showed that the precursor P123 is cleaved with a half-life of approximately 19 min to produce P12 and nsP3; P12 is then cleaved with a half-life of approximately 9 min to produce nsP1 and nsP2. Thus, although the rate of cleavage between nsP1 and nsP2 is faster than that between nsP2 and nsP3, the latter cleavage must occur first and is therefore the rate-limiting step. The rate at which P34 is chased suggests that the cleavage between nsP3 and nsP4 is the last to occur; however the regulation of nsP4 function in Sindbis virus-infected cells may be even more complex than was previously thought. The products nsP1 and nsP2 (and nsP4) are relatively stable; nsP3, however, is unstable, with a half-life of about 1 h, and appears to be modified to produce heterodisperse, higher-molecular-mass forms. In general, the processing schemes used by Sindbis virus and Semliki Forest virus appear very similar, the major difference being that most nsP3 in Sindbis virus results from termination at an opal condon, whereas in Semliki Forest virus cleavage of the P34 precursor is required.     

Preparation of monospecific antibodies against two forms of rat liver cytochrome P-450 and quantitation of these antigens in microsomes.

Antibodies prepared against purified cytochrome P-450 and P-448 from phenobarbital- and 3-methylcholanthrene-pretreated rats have been shown to recognize several forms of hepatic cytochrome P-450 (P. E. Thomas, A. Y. H. Lu, D. Ryan, S. B. West, J. Kawalek, and W. Levin, 1976, Mol. Pharmacol.12, 746–758). These antibodies have been made monospecific for a single form of cytochrome P-450 by immunoadsorption with partially purified solid-phase cytochrome P-450 from rats treated with a different inducer than that used for isolation of the antigen. Each monospecific antibody did not react with different forms of cytochrome P-450 present in the heterologous antigen preparation. These monospecific antibodies, covalently bound to Sepharose, were used to purify the antigens (catalytically inactive) from microsomes in a single step. The high specificity of these antibodies for a single form of cytochrome P-450 was used to quantitate two forms of cytochrome P-450 in rat liver microsomes by radial immunodiffusion. The percentage of the total cytochrome P-450 in microsomes that is represented by each of these two forms of cytochrome P-450 varied from 3 to 89% depending on the xenobiotic pretreatment of the rats.     

Simultaneous triple-immunogold staining of virus and host cell antigens with monoclonal antibodies of virus and host cell antigens in ultrathin cryosections

The mechanism of intracellular maturation and sorting of herpes simplex virus type I glycoproteins is not known in details. To elucidate the intracellular sorting of viral glycoproteins and their possible interaction with the cytoskeleton, a method for simultaneous immunogold staining of three antigens in ultrathin cryosections is described. Each antigen is stained by an indirect technique using mouse monoclonal IgG as first layer, rabbit anti-mouse IgG as second and gold-conjugated goat anti-rabbit IgG as third layer antibody. After each staining cycle the sections are covered by methyl cellulose and exposed to paraformaldehyde vapour at 80 degrees C for 30 min. This destroys the free antigen combining sites of the second and the third layer IgG and abolish contaminating staining. Simultaneous triple-staining is documented with three mouse monoclonal antisera specific for 1) herpes simplex virus type 1 glycoprotein C, 2) glycoprotein D and 3) alpha- and beta-tubulin as primary antibodies. Labelling for virus glycoproteins was found in some Golgi vesicles and close to the cytoplasmic microtubules as well as on the cell surface and on intracytoplasmic and extracellular virus particles.     

Simultaneous demonstration of two antigens in ultrathin cryosections by a novel application of an immunogold staining method using primary antibodies from the same species

A recently developed immunocytochemical double-staining method for ultrathin Epon and Lowicryl K4M sections has been adopted for use on ultrathin cryosections. The essential features of the method include: staining for the first antigen by the indirect method using sufficient concentrations of second antibodies conjugated to colloidal gold particles to saturate available epitopes on the primary antibodies; supporting the cryosections by methyl cellulose followed by paraformaldehyde vapour treatment (30-60 min at 80 degrees C); removal of the methyl cellulose followed by staining for the second antigen using primary antiserum from the same species and another size class of colloidal gold particles conjugated to second antibodies. Contaminating staining does not occur if the paraformaldehyde vapour treatment exceeds 30 min, as this treatment destroys the combining sites on the second antibodies applied in the first staining cycle. Successful double-staining was documented using primary rabbit antibodies to growth hormone and corticotropin and anti-rabbit IgG conjugated to 5 and 15 nm colloidal gold particles. Following double-staining, the ultrathin cryosections may be silver-enhanced to improve detectability of the markers at low magnification.     

Cytoplasmic filaments in the endothelial cells of the sheathed capillary: an ultrastructural and immunocytochemical study in the pig spleen.

Cytoplasmic filaments of the endothelial cells of sheathed capillaries in the pig spleen were identified and their ultrastructure was studied. Two types of cytoplasmic filaments were found: intermediate filaments (diameter: 10 nm) which filled most of the interior of the cells, and thin filaments (diameter: 5 nm) which were located just beneath the cell membrane and filled the lateral cytoplasmic processes. In immunocytochemical preparations, the intermediate filaments were positive for vimentin and desmin, and were negative for keratin. Staining of the thin filaments with heavy meromyosin resulted in arrowhead formations. These observations suggest that the intermediate filaments maintain the cytoarchitecture, possibly protecting the cell from structural alterations induced by blood pressure changes. Concurrently, thin filaments may facilitate the passage of red blood cells and blood platelets through the interendothelial fenestrae of the sheathed endothelial cell to the reticular meshwork in the capillary sheath.     

Common antigens of oval cells and cholangiocytes in the mouse. Their detection by using monoclonal antibodies

Monoclonal antibodies (MAb) were produced against antigens (Ag) of oval cells isolated from the preneoplastic murine liver. To suppress the immune response to major antigens common with hepatocytes, the principle of anti-idiotype immunization was employed. Characteristics of three MAb reacting selectively with the foci of oval cell proliferation are described. MAb A6 and G7 detected two different antigens (Ag A6 and Ag G7, respectively) common for oval cells and cholangiocytes. Ag A6 was also found in normal parenchyma (in membranes of single hepatocytes adjacent to portal veins), in the preneoplastic liver (in hepatocytes formed de novo) and in some hepatoma cells. Ag G7 was not detected in hepatocytes. MAb E5 stained the matrix in the areas adjacent to oval cells and large bile ducts. All the three Ag were widely distributed in normal tissues of mice. The significance of the detected Ag as markers of murine liver epithelial cell lines and stages of their differentiation is discussed as well as the possible relationship between Ag A6 and Ag of human blood groups.     

Double staining to increase the sensitivity of protein detection in polyacrylamide gels.

As more and more researchers are examining proteins that are available only in extremely limited quantities, i.e., cellular extracts or genetic engineering products, it is critical to utilize staining methods that maximize sensitivity. The protocol we describe here--double staining of polyacrylamide electrophoresis gels with Pro-Blue (colloidal blue stain) followed by silver staining--yields an extremely sensitive, nonspecific protein stain. On average, this double-staining technique resulted in a 40-fold increase in sensitivity and intensity vs. silver stain alone. This is a tremendous return for a small investment in additional time and materials.     

Specific in vivo staining of astrocytes in the whole brain after intravenous injection of sulforhodamine dyes

Fluorescent staining of astrocytes without damaging or interfering with normal brain functions is essential for intravital microscopy studies. Current methods involved either transgenic mice or local intracerebral injection of sulforhodamine 101. Transgenic rat models rarely exist, and in mice, a backcross with GFAP transgenic mice may be difficult. Local injections of fluorescent dyes are invasive. Here, we propose a non-invasive, specific and ubiquitous method to stain astrocytes in vivo. This method is based on iv injection of sulforhodamine dyes and is applicable on rats and mice from postnatal age to adulthood. The astrocytes staining obtained after iv injection was maintained for nearly half a day and showed no adverse reaction on astrocytic calcium signals or electroencephalographic recordings in vivo. The high contrast of the staining facilitates the image processing and allows to quantify 3D morphological parameters of the astrocytes and to characterize their network. Our method may become a reference for in vivo staining of the whole astrocytes population in animal models of neurological disorders.     

Characterization of cross-reacting antigens on the Epstein-Barr virus envelope and plasma membranes of producer cells.

A rabbit antiserum has been prepared against the B95-8 transforming strain of EBV. The antiserum has a high virus neutralizing titer (approximately 1:1000) against both the marmoset B95-8 EBV and the human P3HR-1 EBV. The neutralizing antibodies may be absorbed completely with EBV producer cell lines, but not with nonproducer cell lines or producer cell lines treated with phosphonoacetic acid (PAA) so as to be nonproducer. After repeated absorption with PAA-treated B95-8, the serum remains reactive with the membranes of producer cell lines as judged by immunofluorescence or the 125I--Staphylococcal protein A radioimmunoassay. Thus the neutralizing antigens are expressed on the membranes of producer cell lines and may be purified from this source using the serum and 125I--Staph A binding as an assay. The ability of the serum to differentiate between producer and nonproducer cells by means of cell surface determinants has been exploited to achieve a separation of these two populations from the same culture. Immunoprecipitation by the protein A technique shows that the serum recognizes two polypeptides from producer cells of approximate molecular weights 150,000 and 75,000.