Reduction of High Background Staining by Heating Unfixed Mouse Skeletal Muscle Tissue Sections Allows for Detection of Thermostable Antigens With Murine Monoclonal Antibodies

Antigen detection with indirect immunohistochemical methods is hampered by high background staining if the primary antibody is from the same species as the examined tissue. This high background can be eliminated in unfixed cryostat sections of mouse skeletal muscle by boiling sections in PBS, and several proteins including even the low abundant dystrophin protein can then be easily detected with murine monoclonal antibodies. However, not all antigens withstand the boiling procedure. Immunoreactivity of some of these antigens can be restored by subsequent washing in Triton X-100, whereas immunoreactivity of other proteins is not restored by this detergent treatment. When such thermolabile proteins are labeled with polyclonal primary antibodies followed by dichlorotriazinylaminofluorescein–conjugated secondary antibodies and boiled, the fluorescence signal persists, and sections can then be processed with a monoclonal antibody for double immunostaining of a protein unaffected by boiling. This stability of certain fluorochromes on heating can also be exploited for double immunofluorescence labeling of two different thermostable proteins with murine monoclonal antibodies as well as for combination with Y-chromosome fluorescence in situ hybridization. Our method should extend the range of monoclonal antibodies applicable to tissues derived from the same species as the monoclonal antibodies. (J Histochem Cytochem 56:969–975, 2008)     

Immunogold staining method for the light microscopic detection of leukocyte cell surface antigens with monoclonal antibodies: its application to the enumeration of lymphocyte subpopulations

Colloidal gold was used as a marker for the light microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies. Suspensions of peripheral blood leukocytes were first incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. The cells were fixed and cytocentrifuge preparations or smears were made. Granulocytes and monocytes were then labeled by the cytochemical staining of their endogenous peroxidase activity. Lymphocytes reacting with the monoclonal antibody had numerous dark granules around the surface membrane. With electron microscopy, these granules appeared as patches of gold particles. This immunogold staining method proved to be a reliable tool for the enumeration of T-lymphocyte subpopulations in peripheral blood. The results were almost identical to those obtained with immunofluorescence microscopy. The procedure can also be applied on small volumes of capillary blood. This constitutes a good microtechnique for the determination of lymphocyte subsets in children.     

The animal research kit (ARK) can be used in a multistep double staining method for human tissue specimens.

The newly developed Animal Research Kit (ARK) offers a simple and economic way of biotinylating mouse primary antibodies for background-free immunostaining of mouse and rat tissue specimens. Biotinylation involves the use of a biotinylated goat anti-mouse immunoglobulin Fab fragment mixed with a mouse primary antibody and subsequent blocking with normal mouse immunoglobulin. Because a reliable immunoenzyme double staining procedure on human tissue specimens with two unlabeled mouse primary antibodies of identical subclass is almost impossible, we have tested the performance of ARK biotinylation of one primary antibody in a multistep indirect/direct staining protocol. The multistep double staining procedure involved the subsequent application of an unlabeled mouse monoclonal antibody (MAb) 1 detected with an enzyme-labeled EnVision reagent, normal mouse serum for blocking, followed by a biotinylated mouse MAb 2 and enzyme-labeled streptavidin. Alkaline phosphatase and peroxidase enzymatic activities were developed last. Double staining results obtained with an ARK biotinylated reagent were compared with a truly biotinylated reagent using N-hydroxy succinimide-biotin for conjugation. It appeared that both biotinylation procedures revealed identical double staining results. Although a limited number of antibody combinations have been tested, it is clear that this double staining procedure will be successful for many antibody pairs.     

Use of a monoclonal rat anti-mouse Ig light chain (RAMOL-1) antibody reduces background binding in immunohistochemical and fluorescent antibody analysis

Rat monoclonal antibodies (MAb) directed to mouse Ig heavy and light chain determinants were produced. A rat anti-mouse light chain MAb (RAMOL-1) which bound to all (24/24) mouse Ig of the kappa light chain type and with varying strength to 4/4 lambda light chain-bearing Ig was evaluated as a general secondary reagent, together with two MAb that bound to the heavy chain of mouse IgG. They were conjugated with biotin or FITC and used in immunohistochemical and immunofluorescence assays to detect mouse monoclonal antibodies binding to antigens expressed in rat and human tissues and cells. As compared to commercially available polyclonal reagents, RAMOL-1 gave higher staining contrast by showing lower background staining and equal or higher staining of the primary MAb tested. This was a result of two main effects. First, crossreactivity with endogenous Ig and tissue type-specific determinants was eliminated. With polyclonal anti-mouse Ig reagents, binding to endogenous Ig was noted in vascular spaces and on Ig-bearing cells, and to rat gastric mucosa and epithelial tumor tissue in frozen tissue sections, even when diluted in high concentrations of serum homologous to the tissue. Second, binding of the secondary reagent was reduced to cells and tissues prone to have high nonspecific binding capability, such as monocytes/macrophages and formalin-fixed, paraffin-embedded tissue. Owing to unlimited and reproducible access to this homogeneous reagent, RAMOL-1 is used as second antibody to standardize the procedure used for immunohistochemical grading of human malignant tumors by determination of blood group antigen expression detected with mouse MAb.     

One-step double immunolabeling of mouse interdigitating reticular cells: simultaneous application of pre-formed complexes of monoclonal rat antibody M1-8 with horseradish peroxidase-linked anti-rat immunoglobulins and of monoclonal mouse anti-Ia antibody with alkaline phosphatase-coupled anti-mouse immunoglobulins

A novel one-step double immunolabeling method was elaborated on the basis of the simultaneous application of preformed molecular complexes of two primary antibodies with their specific secondary antibodies labeled with different enzymes. Treatment with a rat monoclonal antibody (MAb), M1-8, pre-coupled with horseradish peroxidase-linked sheep anti-rat immunoglobulins, and enzyme reaction revealed by the 3-amino-9-ethylcarbazole/hydrogen peroxide reaction, resulted in red-brown intracytoplasmic staining of interdigitating reticular cells in the lymph nodes of Balb/c mice. Another molecular complex, made of mouse anti-Ia MAb with alkaline phosphatase-linked rabbit anti-mouse immunoglobulins, applied at the same time and then developed with naphthol AS-BI-phosphate/fast blue BB as substrate, yielded blue surface staining of this cell type in addition to labeling of B-lymphocytes. The method described provides the possibility of relatively rapid double antigen detection where the binding sites of the secondary antibodies are saturated by the specific primary immunoglobulins. This approach seems to avoid nonspecific binding of primary antibodies to Fc receptors, and the unwanted binding of secondary antibodies with cell surface immunoglobulins on B-lymphocytes or with crossreactive primary antibodies used in the other sequence, if the primary antibodies and the tissue are the same or crossreactive animal species.     

Background-free Detection of Mouse Antibodies on Mouse Tissue by Anti-isotype Secondary Antibodies

Immunodetection on mouse routinely processed tissue via antibodies raised in mice faces cross-reactivity of the secondary anti-mouse reagents with endogenous immunoglobulins, which permeate the tissue. Various solutions to this problem have been devised and include endogenous Ig block with anti-mouse Fab fragments or directly conjugated primary antibodies. Mouse isotype-specific antibodies, differently from reagents directed against both heavy and light chains, fail to detect endogenous Ig after fixation and embedding, while providing a clean and specific detection system for mouse antibodies on mouse routinely processed tissue.     

Standardizing Immunohistochemistry: A New Reference Control for Detecting Staining Problems

A new standardized immunohistochemistry (IHC) control for breast cancer testing comprises formalin-fixed human epidermal growth factor receptor 2, estrogen receptor, or progesterone receptor peptide antigens covalently attached to 8-µm glass beads. The antigen-coated beads are suspended in a liquid matrix that hardens upon pipetting onto a glass microscope slide. The antigen-coated beads remain in place through deparaffinization, antigen retrieval, and immunostaining. The intensity of the beads’ stain provides feedback regarding the efficacy of both antigen retrieval and immunostaining. As a first report, we tested the sensitivity and specificity of the new IHC controls (“IHControls”). To evaluate sensitivity, various staining problems were simulated. IHControls detected primary and secondary reagent degradation similarly to tissue controls. This first group of IHControls behaved similarly to tissue controls expressing high concentrations of the antigen. The IHControls were also able to detect aberrations in antigen retrieval, as simulated by sub-optimal times or temperatures. Specificity testing revealed that each antigen-coated bead was specific for its cognate IHC test antibody. The data support the conclusion that, like tissue controls, IHControls are capable of verifying the analytic components of an immunohistochemical stain. Unlike tissue controls, IHControls are prepared in large bulk lots, fostering day-to-day reproducibility that can be standardized across laboratories.     

Improvement of Opal Multiplex Immunofluorescence Workflow for Human Tissue Sections

The Opal multiplex technique is an established methodology for the detection of multiple biomarkers in one section. The protocol encompasses iterative single stainings and heating-mediated removal of the primary and secondary antibodies after each staining round, leaving untouched the Opal fluorophores which are deposited onto the antigen of interest. According to our experience, repetitive heating of skin sections often results in tissue damage, indicating an urgent need for milder alternatives to strip immunoglobulins. In this study, we demonstrate that considerable heating-related damage was found not only in skin but also in tissues of different origin, mostly characterized by low cell density. Importantly, the morphology remained fully intact when sections were repetitively exposed to β-mercaptoethanol-containing stripping buffer instead of multiple heating cycles. However, target epitopes appeared sensitive at a differential degree to multiple treatments with stripping buffer, as shown by loss in staining intensity, but in all cases, the staining intensity could be restored by increment of the primary antibody concentrations. Application of β-mercaptoethanol-containing stripping buffer instead of heating for antibody removal markedly improved the quality of the Opal multiplex technique, as a substantial higher number of differently colored cells could be visualized within a well-conserved morphological context:     

Nonlabeled secondary antibodies augment/maintain the binding of primary, specific antibodies to cell membrane antigens.

BACKGROUND: In studies on surface membrane antigen expression using immunofluorescence techniques, it is commonly observed that direct staining gives weaker signals than the signals following indirect staining with fluorochrome-conjugated secondary antibodies. This is most marked when cells have also been permeabilized in order to stain intracellular protein. The commonly accepted explanation for this observation is that fluorochrome-conjugated secondary antibodies bind to a higher number of binding sites on the primary antibody, as compared to the binding of conjugated primary antibodies to the membrane antigens. Another hypothesis might be that the antibody/antibody complexes formed on the membranes when using the indirect technique may have an augmented ability to bind the membrane epitopes. The present study was performed in order to check this hypothesis. MATERIALS AND METHODS: Peripheral blood mononuclear cells were stained with fluorochrome-conjugated anti-CD antibodies directly without or with a second-step application of nonconjugated goat anti-mouse IgG antibodies, followed by different fixation and permeabilization methods. The cells were analyzed by flow cytometry. RESULTS: A second-step application of nonconjugated goat anti-mouse IgG antibodies following direct staining with fluorochrome-conjugated anti-CD antibodies gave a significant increase in membrane antigen expression on permeabilized cells as compared to direct staining alone. The secondary antibody must be bivalent, since whole IgG or F(ab')(2) fragments of the goat anti-mouse antibodies showed effects, while Fab fragments did not. CONCLUSIONS: Nonlabeled secondary antibodies are able to influence the binding of primary, specific antibodies to cell membrane antigens on cells treated with permeabilizing agents necessary for staining intracellular proteins. The improved membrane antigen expression seems to be due to the formation of a network of primary and secondary antibodies on the cell surface, with increased ability for maintaining binding to CD antigens.     

Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific “unmasking” treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman’s membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the “unmasking” did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.     

An indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo- and chlorodeoxyuridine incorporated into DNA

In this paper we describe an indirect fluorescence double staining procedure for the simultaneous detection of IdUrd and CldUrd in the same cell nucleus. Two commercially available antibodies were selected for this purpose. A rat anti-BrdUrd monoclonal antibody from Sera-lab was found to bind specifically to CldUrd and BrdUrd. A mouse monoclonal anti-BrdUrd antibody from Becton Dickinson used in a 1:2 dilution binds to all halogenated deoxyuridines but, when the cells were extensively washed with Tris buffer with a high salt concentration, almost no binding to CldUrd was observed. An immunofluorescence procedure was developed, based on these primary antibodies, raised in different species (rat and mouse), in combination with highly purified second antibodies: FITC conjugated goat antirat and Texas-Red conjugated goat antimouse.     

Use of commercially available monoclonal antibodies for immunoenzyme double staining

Summary An immunoenzyme double-staining method for the simultaneous detection of two cellular epitopes, using commercially available mouse monoclonal antibodies, is described. The method employs a combination of the suppression of endogenous biotin and two successive indirect techniques with a blocking step in between. The first indirect method involves an unlabelled monoclonal antibody followed by an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin. After a blocking step with normal mouse serum, the second indirect method is applied using a biotinylated monoclonal antibody followed by the visualization of this antibody by avidin-biotinylated peroxidase complex (ABC) or rabbit anti-biotin and peroxidase-conjugated swine anti-rabbit immunoglobulin in successive steps. Using these methods in combination with the introduction of dioctyl sodium sulphosuccinate and tetramethylbenzidine as chromogens for peroxidase activity, two cellular epitopes could be distinguished clearly in tissue sections by the green-and violet-stained peroxidase and alkaline phosphatase activities, respectively. The expression of two epitopes on the same cellular constituent is outlined by the coappearance of both enzyme activities as a bluish-purple colour. This method allows for the simultaneous identification, localization and enumeration of two cellular epitopes. These can serve as parameters for a number of pathological processes.     

Production and characterization of monoclonal antibodies to rabbit lymphocyte subpopulations. I. Tissue immunofluorescence and flow cytometric analysis

Nine monoclonal antibodies to rabbit T cells and B subpopulations have been generated from three separate fusions of spleen cells from mice immunized with fractionated populations of rabbit lymphocytes. These monoclonal antibodies, as well as a previously described rabbit T cell monoclonal antibody, 9AE10, have been analyzed by immunofluorescence staining on frozen tissue sections of rabbit thymus, spleen, and appendix. This screening method permits rapid identification of the lymphocyte subdomains in each tissue which is not possible by other screening methods. Each monoclonal antibody selected has a unique tissue staining pattern. Flow cytometric analysis of these monoclonal antibodies, using indirect immunofluorescence techniques on thymocytes, splenocytes, and PBL, revealed varying percentages of positive cells and individual mean fluorescence intensities indicating different epitope densities for each antigen. These monoclonal antibodies are now being used to characterize normal lymphocyte function and the role of specific lymphocyte subpopulations in experimental disease models in the rabbit.     

Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter

One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80^o C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.     

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.     

Abrogation of antibody responses in rats to murine monoclonal antibody 791T/36 by treatment with daunomycin-cis-aconityl-791T/36 conjugates

Summary The majority of monoclonal antibodies in clinical use are of murine origin. It is now well-established that patients generate an antibody response to the mouse immunoglobulin which restricts repeated administration. Pre-sensitization of patients to mouse antibody is screened by hypersensitivity to i.d. administered antibody. This study shows that low doses of mouse antibody administered either i.d. or s.c. are highly immunogenic and suggests that a serological assay would be a safer method of screening for anti-mouse antibodies. Rats treated with monoclonal antibody linked via an acid labile cis-aconityl bond to daunomycin failed to produce a primary response to this conjugate. They were also rendered immunologically unresponsive to subsequent challenges with the unconjugated monoclonal antibody. The induced state of immunological unresponsiveness to free antibody persisted in the rats for 18 weeks and although antibody-cis-aconityl-daunomycin pre-treated animals eventually responded to the fourth challenge with free antibody, at week 25, the response was still significantly less than in the free antibody-pre-treated and challenged animals. These studies show that the use of antibody-cis-aconityl-duanomycin conjugates may provide an approach for the control of human responses to mouse immunoglobulin.     

Characterization of Antibodies to Products of Proinsulin Processing Using Immunofluorescence Staining of Pancreas in Multiple Species

The efficient processing of proinsulin into mature insulin and C-peptide is often compromised under conditions of beta cell stress, including diabetes. Impaired proinsulin processing has been challenging to examine by immunofluorescence staining in pancreas tissue because the characterization of antibodies specific for proinsulin, proinsulin intermediates, processed insulin and C-peptide has been limited. This study aimed to identify and characterize antibodies that can be used to detect products of proinsulin processing by immunofluorescence staining in pancreata from different species (mice, rats, dog, pig and human). We took advantage of several knockout mouse lines that lack either an enzyme involved in proinsulin processing or an insulin gene. Briefly, we report antibodies that are specific for several proinsulin processing products, including: a) insulin or proinsulin that has been appropriately processed at the B-C junction; b) proinsulin with a non-processed B-C junction; c) proinsulin with a non-processed A-C junction; d) rodent-specific C-peptide 1; e) rodent-specific C-peptide 2; and f) human-specific C-peptide or proinsulin. In addition, we also describe two ‘pan-insulin’ antibodies that react with all forms of insulin and proinsulin intermediates, regardless of the species. These antibodies are valuable tools for studying proinsulin processing by immunofluorescence staining and distinguishing between proinsulin products in different species.     

Color-Contrast Staining of Two Different Lymphocyte Subpopulations: A Two-Color Modification of Alkaline Phosphatase Monoclonal Anti-Alkaline Phosphatase Complex Technique

A dual staining method for different human lymphocyte subpopulations with nonoverlapping antigen distribution patterns is described. Cytocentrifuge slide preparations of peripheral blood nonadherant mononuclear cells (NAMNC), bone marrow aspirate or buffy coat smears were fixed in acetone and incubated with a primary mouse monoclonal antibody (MAb) against a lymphocyte antigen (CD8, Ig-light-chain, CD19, CD4) followed by rabbit anti-mouse immunoglobulin (Ig) and the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) complex. After repeating the “bridge” antibody and the APAAP, a red product was developed with fast red TR-naphthol AS-BI phosphate. Following this one-color stain the process was repeated using a different primary mouse MAb against another lymphocyte antigen (CD4, Ig-light chain, CDS, MHCU DR, CD5) and fast blue BB-naphthol AS-MX phosphate at the last step to yield a blue product. Control slides stained by the standard one-color APAAP method with die relevant primary MAb showed that there was no nonspecific labelling and the percent of positive cells in a given test was almost identical. To achieve an intense blue in the second stain for some antigens, e.g., CD4, either the MAb concentration had to be increased or two different MAbs recognizing differing epitopes of the same antigen, e.g., T1 and UCHT2 for CD5, were applied. Any change of red to purple at the site of the first stain after 15 min exposure to the blue-yielding AP substrate is due to residual AP activity of the first stain rather than to crossbinding of immunoreagents. This rechnique allows sensitive two-color staining without being limited to the few antigens against which heteroantibodies are available. High nonspecific background staining due to endogenous enzyme activity, as inherent in peroxidase staining procedures, is overcome in this dual APAAP technique.     

Quantitative immunofluorescence, anti-ras p21 antibody specificity, and cellular oncoprotein levels

A general approach to investigating specificity and saturation of antibodies by quantitative immunofluorescence is applied to monoclonal antibodies generated against p21 or ras oligopeptides to quantify ras p21 oncoprotein in cultured cells. Ras 10, a panreactive mouse monoclonal antibody, appears to be a superior probe for detection of p21 in cell extracts or fixed cells because it binds a 21 kD protein on SDS-PAGE/western blots and labels the cytoplasmic membrane in a saturable and competitive manner. RAP-5, a widely used mouse monoclonal antibody generated against an oligopeptide of ras p21, does not recognize p21 in denaturing immunoblots or in immunofluorescence of cultured cells.     

A novel modification of the avidin-biotin complex method for immunohistochemical studies of transgenic mice with murine monoclonal antibodies.

When mouse tissues are probed with murine monoclonal antibodies (MAb) by indirect immunohistochemistry, the secondary antibody detects tissue-bound MAb and irrelevant, endogenous mouse immunoglobulins. The latter are a source of confounding background, especially in diseased tissues. To circumvent this problem, we generated complexes of primary MAb and biotinylated secondary antibodies in vitro for use as antigen-specific probes. After blocking free binding sites in the complexed secondary antibodies with normal mouse serum, the complexes were applied to mouse tissue sections and tissue-bound complexes were visualized with an avidin-biotin detection system. Complexes formed with 12 different rat or mouse MAb were used to probe sections of normal mice, tumor-bearing transgenic mice, and mice with tumor xenografts. The staining patterns produced by these probes reflected the specificity of the MAb in the complexes, and the labeling of irrelevant, endogenous mouse immunoglobulins was reduced substantially. This novel, indirect immunohistochemical method can be exploited to study normal and diseased mouse tissues using a variety of murine MAb.