Multiple immunoenzyme staining: methods and visualizations for the observation with spectral imaging.

Several staining concepts and color combinations exist to perform successful double immunoenzyme staining on human tissue specimens. Most of these concepts are based on differences between both primary antibodies: animal species, mouse Ig isotype or IgG subclasses, conjugates, or concentrations. Traditionally, double immunoenzyme staining has used chromogens selected to provide maximum color contrast when observed with the unaided eye. Unfortunately, visually good color combinations always include at least one diffuse chromogen, because of the paucity of appropriate chromogen colors. This situation is drastically changed with the use of spectral imaging, where multicolor microscopy can be unmixed in individual images based on their spectral characteristics. Spectral unmixing can be performed even up to quadruple immunoenzyme staining. This work contains practical suggestions for immunoenzyme double staining procedures for some frequently encountered primary antibody combinations: rabbit-mouse, goat-mouse, mouse-mouse, and rabbit-rabbit. The suggested protocols are all suitable for a classical red-brown color combination plus blue nuclear counterstain that is composed of peroxidase activity (diaminobenzidine tetrahydrochloride), alkaline phosphatase activity (Liquid Permanent Red), and hematoxylin, respectively. Although the red and brown chromogens do not contrast very well visually, they both show a crisp localization and can be perfectly unmixed by spectral imaging.     

Combined beta-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situ hybridization.

A combination of beta-galactosidase enzyme and the immunogold/silver staining method was studied for evaluation of double-staining experiments. Applications are shown for immunohistochemical double staining using two monoclonal antibodies and for combined immunohistochemistry and DNA in situ hybridization in one tissue section. The following advantages for the present double-staining method were evaluated: superior sensitivity of the immunogold/silver staining method for at least one epitope, which also allows detection of biotinylated DNA probes. The structure of the indolyl precipitate after revelation of beta-galactosidase activity did not show a concealing effect during a sequential double-staining method, as compared with the visualization of peroxidase with diaminobenzidine. These factors, and the sharply contrasting colored reaction products of beta-galactosidase (blue-green) and the immunogold/silver staining method including silver enhancement (brown-black), allow clear distinction of mixed-stained cell constituents.     

Simultaneous immunoenzyme staining of vimentin and cytokeratins with monoclonal antibodies as an aid in the differential diagnosis of malignant mesothelioma from pulmonary adenocarcinoma

The occurrence and coexpression of the cytoskeletal proteins vimentin and cytokeratins were studied in malignant mesotheliomas and pulmonary carcinomas. For this purpose a double immunoenzyme staining with monoclonal antibodies was developed which made it possible to visualize vimentin and cytokeratins simultaneously within the same cell. A clear distinction between stromal cells (vimentin only) and tumour cells was also obtained. A total of 12 mesotheliomas (six mixed type and six epithelioid type) and 13 carcinomas (eight adenocarcinomas and five large cell undifferentiated carcinomas) were studied. The results revealed a clear difference between mesotheliomas and adenocarcinomas: 11 of 12 mesotheliomas showed coexpression of vimentin and cytokeratins in at least 50% of the tumour cells, while in seven of the eight adenocarcinomas none or only a few cells could be seen with this coexpression. In the undifferentiated large cell carcinomas three of five expressed both components, but in less than 25% of the cells. It is concluded that a reliable double immunoenzyme staining of vimentin and cytokeratins can be used as an additional means to distinguish malignant mesothelioma from pulmonary adenocarcinoma.     

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.     

An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide

A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.     

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.     

Simultaneous enumeration of T-cell subsets and macrophages in bronchoalveolar lavage fluids by immunoenzyme double staining. Comparison with conventional immunofluorescence

Bronchoalveolar lavage is a common research and clinical tool for the retrieval of cells from the lower respiratory tract. In addition to conventional morphologic study of these cells, the subtyping of T lymphocytes is often important for reaching a diagnosis of a disease or assessing its activity; subtyping is usually done by a standard immunofluorescence assay on cell suspensions requiring about 5 X 10(5) cells. Since the number of leukocytes in the lavage fluid from many patients is too small to obtain reliable information by this assay, a double immunoenzyme staining of T-lymphocyte subtypes on Cytospin preparations was utilized. This method, which requires a small number of cells, was compared with the standard immunofluorescence assay for the identification and quantitation of lymphocyte subtypes in the lavage fluids of patients with different disorders. Although the immunoenzyme double staining assay is somewhat more laborious, it provides important advantages: (1) simultaneous observation of two lymphocyte subsets and macrophages on the same slide; (2) a considerably smaller number of cells (2 X 10(4) instead of 5 X 10(5] is necessary; (3) the availability of permanent preparations; (4) the possibility of storing the Cytospin slides before staining; and (5) conventional light microscopy can be used. Since the reliability of both techniques appeared to be the same, the double staining assay for routine usage with bronchoalveolar lavage fluids appears to be preferable.     

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections.

Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehyde-fixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.     

The limitations of immunoenzyme approaches to distinguish between 'specific' and 'non-specific' antibody-forming cells, with particular respect to immunocytochemical studies on the in situ immune response

After antigenic stimulation, specific antibody-forming cells can be detected in situ in tissue sections of lymphoid organs using an antigen specific immunoenzyme approach. Immunoglobulin-forming cells staining positively with a particular antigen-enzyme conjugate are considered to be specific antibody-forming cells (provided that the right control conjugates show a negative staining). Immunoglobulin-forming cells, staining negatively with the same antigen-enzyme conjugate, may do so for two reasons: (i) they are in fact specific antibody-forming cells, but are not detected as such because of limitations of the technique; (ii) they are real-nonspecific immunoglobulin producing cells. In this paper, the limitations of the technique are discussed.     

Direct detection of herceptin/trastuzumab binding on breast tissue sections.

The protooncogene product HER-2/neu is the target of the humanized monoclonal antibody trastuzumab (Herceptin). Several tests are used clinically to identify patients with HER-2/neu overexpression based on evaluation by pathologists of gene amplification by fluorescence in situ hybridization or protein expression using immunohistochemistry (IHC). A simple technique has been developed for staining formalin-fixed, paraffin-embedded breast cancer tissue using unmodified Herceptin/trastuzumab as the primary antibody. Results were compared with staining with the commercial kit, HercepTest, as well as with polyclonal anti-HER-2/neu antibodies and with biotinylated trastuzumab. These procedures were tested using four breast cancer microarrays. There were 854 cores that were stained with all four antibodies, representing 325 cases. A standard 4-point scoring system (0-3) was used. A total of 156 cases (48%) were scored as 0 by all the methods used and 31 (9.5%) were positive (3+) by all methods. Of interest, three cases scored negative using polyclonal anti-HER-2/neu antibodies but were positive using unmodified trastuzumab. To clarify this discrepancy, whole sections of tumors were examined with both antibodies using double labeling. There were some tumors that demonstrated a mosaic pattern of staining with neighboring cells or groups of cells stained exclusively with one antibody or the other. These results demonstrate that unmodified humanized or human therapeutic antibodies could be used for preclinical testing or in a clinical laboratory setting for IHC-based selection of patients for treatment, and results of such selection could be different from those obtained using polyclonal antibody-based IHC procedure.     

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections

Summary Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehydefixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.     

Transforming growth factor-alpha expression in benign and malignant human prostatic disease.

Investigation of biological variables in prostatic disease may not only prevent patients with a good prognosis being overtreated, but allow better selection of appropriate therapy, and may identify potential targets for novel therapies. This study investigates the growth factor transforming growth factor-alpha (TGF alpha) expression in benign and malignant prostatic biopsies using both radioimmunoassay and immunohistochemistry, considering its role in malignant epithelial transformation and as a prognostic indicator. Biochemical methods were less satisfactory than the more selective immunohistochemical methods, due to the heterogeneity of prostatic tissue. Seventy-one percent of benign biopsies (range 0-18.62ng/mg DNA) and 69% of malignant biopsies (range 0-11.1ng/mg DNA) had detectable levels of TGF alpha using radioimmunoassay. Immunohistochemical staining for TGF alpha identified expression in 15% of benign (4 out of 27) and 53% malignant biopsies (18 out of 34). Positive staining was also identified in premalignant lesions and within stromal elements, thus implying the factor's role in autocrine/paracrine growth and/or malignant transformation. Immunostaining for TGF alpha may enhance detection of premalignant lesions and small foci of malignant glands which are otherwise difficult to identify using standard histopathological techniques.     

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.     

Standardization of various applications of methacrylate embedding and silver methenamine for light and electron microscopy immunocytochemistry

The use of butyl-methyl-methacrylate embedding and the application of the silver methenamine (SM) method as a poststaining of the immunoperoxidase-DAB (IP) procedure led to the standardization of several useful methods for the visualization of tissue antigens at the light and electron microscope level. These procedures included: 1) Standardization of the actual methacrylate embedding; 2) The IP-SM method with and without periodic acid oxidation, which provided 100% intensification of the IP staining; 3) The IP-SM method made it possible to stain semithin sections (0.5 micron), and this in turn, permitted a) clear visualization under the light microscope of the intracellular distribution of antigens and, b) staining, in several adjacent sections, of roughly the same cytoplasmic region of the same cell with different primary antisera; 4) a double immunostaining whereby the first antigen in the sequence was revealed by the IP-SM method and the second by the IP procedure; 5) standardization of the IP and the IP-SM methods for post-embedding staining of ultrathin methacrylate sections. The combined application of methacrylate embedding and the IP-SM, and the use of an appropriate fixative, resulted in an ultrastructural immunocytochemical procedure characterized by a good immunoreactivity of the tissue sections, a strong and selective immunoreaction and a well preserved ultrastructure.     

Standardization of various applications of methacrylate embedding and silver methenamine for light and electron microscopy immunocytochemistry

Summary The use of butyl-methyl-methacrylate embedding and the application of the silver methenamine (SM) method as a poststaining of the immunoperoxidase-DAB (IP) procedure led to the standardization of several useful methods for the visualization of tissue antigents at the light and electron microscope level. These procedures included: 1) Standardization of the actual methacrylate embedding; 2) The IP-SM method with an without periodic acid oxidation, which provided 100% intensification of the IP staining; 3) The IP-SM method made it possible to stain semithin sections (0.5 m), and this in turn, permitted a) clear visualization under the light microscope of the intracellular distribution of antigens and, b) staining, in several adjacent sections, of roughly the same cytoplasmic region of the same cell with different primary antisera; 4) a double immunostaining whereby the first antigen in the sequence was revealed by the IP-SM method and the second by the IP procedure: 5) standardization of the IP and the IP-SM methods for post-embedding staining of ultrathin methacrylate sections.The combined application of methacrylate embedding and the IP-SM, and the use of an appropriate fixative, resulted in an ultrastructural immunocytochemical procedure characterized by a good immunoreactivity of the tissue sections, a strong and selective immunoreaction and a well preserved ultrastructure.Supported by Grant RS-82-18 from Dirección de Investigaciones, Universidad Austral de Chile, Chile     

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.     

Applications of immunocolloids in light microscopy. IV. Use of photochemical silver staining in a simple and efficient double-staining technique

We report the development of a new light-microscopic double-staining technique using colloidal gold as sole marker. The contrasting color to the red of colloidal gold is achieved by the application of photochemical silver reaction. The silver reaction, which is principally performed at the end of the first staining sequence, converts the red color of a gold-labeled reagent into black. This contrasts clearly with the red coloration that results from the second incubation sequence without silver reaction. For antigen double staining, the same protein A-gold complex can be used to provide the black and the red color, thus rendering the technique very economical. Alternatively, combination of protein A-gold immunolocalization and lectin-gold staining is possible, as is combined lectin-gold staining.     

The relevance of various tests for the study of specificity in immunocytochemical staining: a review

Factors determining the specificity of immunocytochemical (ICC) tissue stainings as well as the various tests to study these factors are discussed. Since every specificity test only deals with particular aspects of the ICC procedure, a practical sequence of known test methods is proposed, which enables the determination of the specificity of the ICC tissue staining and, after possibly needed antiserum purification steps, may result in a monospecific staining. It is made clear that such a sequence has always to include a tissue-spectrum affinity test, in which the spectrum of tissue antigens is controlled for antibody binding. A variety of such tests, consisting of separation of tissue compounds, fixation, and ICC detection, are discussed as well as their pros and cons with respect to their predictability for the actual serum specificity in the tissue section.     

A one-day double-labelling technique for tissue specimens: Immunogold-silver staining forin situ hybridization combined with alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry for antigens

Summary An improved technique is described that addresses the problems of sensitivity, specificity, the use of hazardous radioactive equipment and time consumption in immunohistochemical labelling and double labelling ofin situ hybridization of tissue specimens. It consists of a two-step protocol in which digoxigenin-uridine triphosphate (UTP) labelled riboprobes in thein situ hybridization step are visualized by the immunogold-silver staining method, and double labelling of tissue antigens is achieved by the application of an alkaline phosphatase-anti-alkaline phosphatase staining step. We tested this protocol using snap-frozen tissue sections of synovial tissue from patients with rheumatoid arthritis. The target mRNA was detected by perforin or cathepsin D riboprobes, the double labelling was performed using anti-collagen type IV and alpha-smooth muscle actin antibodies. It is concluded that, in comparison with an established three-to four-day double-labelling protocol used in many laboratories, this one-day combination is currently the most rapid assay of reliable quality for double labelling ofin situ hybridization products and tissue antigens.     

Quantitative double-staining methods for tryptophyl and cysteinyl residues and protein in model films

Summary The reliability of three new staining methods developed by the authors were examined with polyacrylamide gel films containing concanavalin A and ovalbumin. The methods were a 2-nitrophenylsulphenyl chloride technique for staining tryptophyl and cysteinyl residues together, a nitrophenylsulphenyl-2-mercaptoethanol method for tryptophyl residues only, and the combination of both these techniques with Coomassie Brilliant Blue for staining total protein. The relative contents of tryptophyl and cysteinyl residues of concanavalin A and ovalbumin in the films, calculated from absorbance values at 370 and 650 nm of double-stained films, were in good agreement with the theoretical values calculated from their amino acid compositions. Thus, these quantitative double-staining methods are reliable and should be useful for histochemical investigations of changes in the amount and composition of tissue proteins.