Double immunocytochemical staining in the study of antibody-producing cells in vivo. Simultaneous detection of cells producing monospecific antibodies and cells producing cross-reacting antibodies in the spleen of rabbits after injection of two related antigens

Rabbits were injected simultaneously with both human gamma globulin (HGG) and bovine gamma globulin (BGG). Sections of spleen tissue were prepared from spleen biopsies taken during the primary or secondary immune response, and incubated simultaneously with horseradish peroxidase (HRP)-HGG conjugate and alkaline phosphatase (AP)-BGG conjugate in order to detect cells containing specific antibodies against one or both of the antigens. After both HRP and AP cytochemistry, cells with a red-stained cytoplasm, cells with a blue-stained cytoplasm, and cells with a violet-stained cytoplasm were detected in the spleen. The red-stained cells had bound the HRP-HGG conjugate, indicating that these cells contained anti-HGG antibodies. The blue-stained cells had bound the AP-BGG conjugate, indicating that these cells contained anti-BGG antibodies. The violet-stained cells had obviously bound both the HRP-HGG conjugate and the AP-BGG conjugate, indicating that these cells contained antibodies cross-reacting with both antigens. Results are compared with earlier studies on the antigenic similarities and differences between HGG and BGG when used as antigens in rabbits.     

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 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.     

A trinitrophenyl(TNP)-poly-L-lysine-horseradish peroxidase conjugate for the detection of anti-TNP antibodies in vivo

A new conjugate for the detection of anti-trinitrophenyl(TNP) antibodies was developed to study the localization pattern of specific antibody containing cells and extracellular antibody in vivo. By means of a bridging molecule, poly-L-lysine, nine TNP groups and six horseradish peroxidase (HRP) groups were joined in one conjugate. Thus a higher specificity (more hapten) was united with a higher staining intensity (more enzyme) in the same conjugate. This conjugate made possible the simultaneous detection of anti-TNP antibody containing cells and establishment of their class (immunoglobulin M (IgM) and IgG). It was also used for the demonstration of anti-TNP antibodies in tissues where a TNP-alkaline phosphate (AP) conjugate could not be used due to high AP (endogenous) background staining. Thus we demonstrated anti-TNP antibody containing cells in gut associated lymphoid tissue and anti-TNP-(TNP-ovalbumin) immune complexes in the glomeruli of the kidney. We suggest that poly-L-lysine is a suitable bridging molecule for the preparation of hapten-HRP conjugates.     

Recent advances in the detection and characterization of specific antibody-forming cells in tissue sections

Summary A new general approach has been developed for the detection of one or more different specific antibody producing cells and the simultaneous determination of their Ig isotype in tissue sections, after immunization of animals. Specificity of intracellular antibodies is demonstrated after incubation of the sections with an antigen-enzyme conjugate and the isotype of the antibodies is determined using an anti-immunoglobulin (Fc chain-specific)-enzyme conjugate followed by histochemical revelation of the two different enzymes. The principles of the method, the required antigen— and antibody—enzyme conjugates and their application in single, double or triple staining studies are reviewed.The method allows the detection of specific antibody-forming cells against protein antigens as well as against haptens. By means of haptens such as trinitrophenyl (TNP), immune responses against thymus dependent, thymus independent, and particulate antigens can be studied. In a limited number of cases the method can also be used to study the localization of antigen—antibody complexes.     

An immunoenzyme triple-staining method using both polyclonal and monoclonal antibodies from the same species. Application of combined direct, indirect, and avidin-biotin complex (ABC) technique

For immunohistological analysis, simultaneous detection of multiple cellular epitopes, as compared to single staining of serial sections, is sometimes needed. Therefore, immunoenzyme triple-staining protocols were tested with polyclonal and monoclonal antibodies on tissue sections and cytospin preparations. Various immunoconjugates were used in different combinations of methods, of which not all proved to be suitable. Of the tested protocols, one yielded superior results for both monoclonal and polyclonal antibodies, with optimal preservation of their original avidity. The method consists of a combination of indirect, direct, and avidin-biotin complex technique. The three antigens can be distinguished clearly and selectively by the reaction products of the enzyme activities of beta-galactosidase (green), alkaline phosphatase (blue), and horseradish peroxidase (red).     

Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry

Summary Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods.In honour of Professor P. van Duijn     

Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry

Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods.     

Flow cytoenzymology of intracellular tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRACP) is a cytochemical marker for hairy cell leukemia, macrophages, dendritic cells, and osteoclasts. Our purpose was to develop multicolor cytofluorometric methods to evaluate intracellular TRACP enzymic activity using a fluorogenic cytochemical reaction in combination with immunochemical stains for distinct surface membrane antigens. Monocyte-derived dendritic cells (DCs) were the model TRACP-expressing cells studied. Intracellular TRACP activity was disclosed using naphthol-ASBI phosphate as substrate with fast red-violet LB salt as coupler for the reaction product. Before the TRACP enzymic reaction, surface antigens, CD86 and CD11c of DCs, were bound with specific fluorescent antibodies to test compatibility of surface labeling and intracellular staining. TRACP activity varied in DCs from donor to donor but was reproducible on repeated examinations of each sample. Samples could be stained for simultaneous analysis of surface antigens and intracellular TRACP activity, provided certain technical details were observed. The TRACP reaction time should not exceed 9 min and the cell number should not exceed 2 x 10(5)/100 micro l test. Fluorescent surface labels did not affect the intensity of the TRACP stain, but the intensity of some surface labels may be diminished by elution of low-affinity antibodies during the TRACP reaction. Readjustment of the threshold settings in triple-labeled cells is needed to compensate for this phenomenon. Intracellular TRACP activity can be quantitated in subpopulations of cells within mixed cell populations by flow cytofluorometry using simple cytochemical methods in combination with fluorescent antibodies to cell-surface and other differentiation antigens. The cytochemical method should be useful for basic investigations of differentiation, maturation, and function of macrophages, DCs, and osteoclasts, and for diagnosis and management of hairy cell leukemia.     

Detection of lipids in the plant meristematic cell with the aid of Sudan black staining

As lipids can be a source of artefacts during intracellular localization of enzymes by cytochemical methodsin situ it was the aim of the present work to obtain orientation data on the distribution of lipids in the meristematic plant cells. The different fixation and object embedding methods examined revealed that it is best to fix the material by some formol fixative and without chroming, to embed it in polyethyleneglycol media. An alcoholic solution of Sudan black was found to be most reliable. In the meristematio cells the cytoplasm is usually stained more intensely than the nucleus. The ground cytoplasm is stained weakly while cytoplasmic particles are stained intensely. In some cases an intense black staining of nuclei, particularly in the prolongation zone, can be achieved. The staining intensity of cell components does not decrease on extracting lipids with pyridine. After extracting the dye from stained cell components a browninsh residual coloration remains. Chromatography of Sudan black revealed in all the samples tested slowly moving spots (blue and violet) and rapidly moving ones (red II, yellow, red I, colourless).     

Simultaneous Formalin Fixation and Edta Decalcification, With Carbowax Embedding for Preservation of Acid Phosphatase

Carbowax serial sections from pubic symphyses of female mice, fixed and decalcified in a 10% formalin-5% Versenate solution for 18 hr at 4 C, pH 5.2, were incubated for 30 min with Burstone's simultaneous coupling reagent (pH 5.2); substrate: naphthol AS-TR and the diazonium salt, fast red violet L.B. All sections were counterstained with 1% methyl green at pH 4.0 in a phospho-citrate buffer. Inhibition by 0.01 M NaF, 0.0002 M CuCl₂, 10% tartaric acid and 0.01 M NaCN, as well as substrate-deficient and heat-inactivated controls, demonstrated conclusively that acid phosphatase was functionally preserved. Strong enzymatic activity was exhibited by osteoclasts, chondroclasts and free multinucleated giant cells. In addition, megakaryocytes, histiocytes, plasma cells, and monocytes exhibited moderate activity. The results demonstrated the technique to be consistently reproducible.     

Detection of endogenous and antibody-conjugated alkaline phosphatase with ELF-97 phosphate in multicolor flow cytometry applications

BACKGROUND: The fluorogenic alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF(R)-97 phosphate, for Enzyme-Labeled Fluorescence) has been used primarily in microscope-based imaging applications to detect endogenous AP activity, antigens and various ligands in cells and tissues, and nucleic acid hybridization. In a previous study, we demonstrated the applicability of ELF-97 phosphate for detecting endogenous AP activity by flow cytometry. In this study, we show that the spectral characteristics and high signal-to-noise ratio provided by the ELF-97 phosphate make it a useful label for immunodetection via flow cytometry. It can be combined with a variety of other fluorochromes for multiparametric flow cytometry analysis of both endogenous AP activity and intracellular and extracellular immunolabeling with AP-conjugated antibodies. METHODS: ELF-97 phosphate detection of endogenous AP activity in UMR-106 rat osteosarcoma cells was combined with intracellular antigen detection using Oregon Green 488 dye-conjugated secondary antibodies and DNA content analysis using propidium iodide (PI) or 7-aminoactinomycin D (7-AAD). ELF-97 phosphate detection of endogenous AP was also tested for spectral compatibility with a variety of other commonly used fluorochromes. ELF-97 phosphate was then used to directly label intracellular antigens via AP-conjugated antibodies, again combined with the analysis of DNA content using PI and 7-AAD. ELF-97 phosphate was also used to directly detect extracellular antigens. It was combined with Oregon Green 488 dye, phycoerythrin (PE), and PE-Cy5 dye-labeled antibodies for simultaneous four-color analysis. All samples were analyzed on a dual-beam flow cytometer, with UV excitation of the ELF-97 alcohol reaction product. RESULTS: Application of the ELF-97 phosphate to detect AP was found to be compatible with immunodetection and DNA staining techniques. It was also spectrally compatible with a variety of other fluorochromes. Endogenous AP activity could be detected simultaneously with both intracellular antigens labeled using Oregon Green 488 dye, PE, Cy5 dye and Alexa Fluor 568 dye-conjugated antibodies, and DNA content analysis with PI or 7-AAD. This multiparametric assay accurately delineated the distribution of AP in cycling cells and was able to identify cell subsets with varying endogenous AP levels. The ELF-97 alcohol reaction product was found to be an effective label for intracellular antigen immunolabeling with AP-conjugated reagents, and could also be combined with PI and 7-AAD. ELF-97 phosphate was also found to be a useful label for extracellular antigen immunolabeling with AP conjugates, and was compatible with Oregon Green 488 dye, PE, and PE-Cy5 dye-labeled antibodies for four-color surface labeling with minimal spectral overlap and color compensation. CONCLUSIONS: ELF-97 phosphate was shown to be a useful label for both endogenous and antibody-conjugated AP activity as detected by flow cytometry. Its spectral characteristics allow it to be combined with a variety of fluorochromes for multiparametric analysis. Cytometry 43:117-125, 2001. Published 2001 Wiley-Liss, Inc.     

Production of monomeric antigen-enzyme conjugate to study requirements for follicular immune complex trapping

Summary Studies concerning the localization of immune complexes in lymphoid follicles and the involvement of these trapped immune complexes in the regulation of the immune response have thus far been performed with poorly defined complexes in terms of size and composition. For that reason, the minimum requirements for trapping in terms of number of antigen- and antibody molecules present in immune complexes could not be determined. We here describe the production and in vivo use of a monomeric HSA-HRP antigen-enzyme conjugate, readily demonstrable in cryostat sections and ELISA. This conjugate was obtained by combining the glutaraldehyde coupling-method with chromatography to fractionate monomeric and multimeric constituents.SDS-PAGE analysis showed that the conjugate consisted of a single molecular species of 109 kDa, whereas the often used periodate oxidation coupling method yielded a heterogeneous population of multimeric, oligomeric and monomeric molecules.We investigated the minimal size requirements for the composition of immune complexes to be trapped in murine spleen follicles using three different conjugates (monomeric HSA-HRP, multimeric HSA-HRP and multimeric HSA-HRP-Penicillin) and a panel of anti-HSA and anti-Penicillin monoclonal antibodies. We demonstrate that the smallest immune complexes, consisting of one antibody and two conjugate molecules, do not localize in splenic follicles. Immune complexes prepared with a single monoclonal antibody localize in follicles only if the epitope recognized occurs repeatedly on the antigen.The relevance of these results for physiological follicular trapping of protein antigens is discussed. The described method for the production of monomeric enzyme-labelled protein applicable in histochemistry and ELISA should prove useful to prepare other conjugates of defined size for studies of trapping and other applications.Abbreviations FDC follicular dendritic cell - HRP horseradish peroxidase - HSA human serum albumin - HY anti-HSA hyperimmune serum - MAb monoclonal antibody - Pen penicillin     

Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates

Three-color fluorescence detection methods are described based upon covalently coupling the dye 2-methoxy-2,4-diphenyl-2(2H)-furanone (MDPF) to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using alkaline-phosphatase-conjugated reporter molecules in combination with the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO-phosphate) as well as horseradish peroxidase-conjugated reporter molecules in combination with the new fluorogenic substrate Amplex Gold reagent. This results in all proteins in the profile being visualized as fluorescent blue signal, those detected specifically with the alkaline phosphatase conjugate appearing as fluorescent red signal and those detected specifically with the horseradish peroxidase conjugate appearing as fluorescent yellow signal. Using conventional secondary antibodies, two different targets may be identified as long as primary antibodies generated from two different species are used in the analysis. However, Zenon antibody labeling technology eliminates this restriction, permitting the simultaneous use of two different mouse monoclonal antibodies or two different rabbit polyclonal antibodies in the same electroblotting experiment. The trichromatic detection system is broadly compatible with UV epi-illuminators combined with photographic or charge-coupled device (CCD) cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. Alternatively, the enzyme conjugates may be detected using a laser-based gel scanner. The trichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of two different target proteins relative to the entire protein profile on a single electroblot, precluding any requirement for running replicate gels that would otherwise require separate visualization of total proteins and subsequent alignment with multiple chemiluminescent or colorimetric signals generated on different electroblots.     

Polarity in the transcytotic processing of apical and basal membrane-bound peroxidase-polylysine conjugates in MDCK cells.

A conjugate of horseradish peroxidase (HRP) to poly(L-lysine) (PLL) was used to characterize a non-lysosomal proteolytic compartment in the MDCK Strain I epithelial cell line. This compartment is expressed in a polar fashion, and is capable of degradation of the PLL moiety in the conjugate followed by release of HRP via a basal-to-apical, but not apical-to-basal, transcytotic pathway. This uptake, cleavage, and transport process appears to require approximately 2 hr, as there is a 2 hr lag-time between conjugate administration to the basal surface and HRP release to the apical medium. Monensin (10 microM) failed to inhibit this process, indicating that participation of the trans-Golgi network (TGN) in the trafficking of internalized conjugate is not the rate-determining step. Inhibition of HRP transport was found to be elicited by 50 micrograms/ml leupeptin, but only when applied to the basal surface. Brief trypsinization of either the basal or apical surfaces of cells preloaded with HRP conjugate showed no appreciable inhibitory effect on the apical release of HRP, indicating that an intracellular compartment rather than surface-bound enzymes is responsible for the degradation of the PLL moiety in the conjugate. Our results demonstrate the presence of an intracellular proteolytic compartment which is accessible in the basal-to-apical, but not apical-to-basal, transport pathway; and this compartment can be exploited for the transcytosis of membrane-bound molecules.     

Cytochemical identification of turbot myeloperoxidase-positive granulocytes by potassium iodide and oxidized pyronine Y staining

Cytomorphological and cytochemical staining are important methods for the identification of cell types, in particular in fish which often lack biological tools such as specific antibodies. Myeloperoxidase (MPO) is usually used as an intracellular marker of neutrophil accumulation in tissues and a marker of neutrophil activity in plasma. In this study, we reported a potassium iodide and oxidized pyronine Y (KI-PyY) staining method for rapid and highly sensitive detection of MPO-positive cells in turbot blood, peritoneum, and tissues. MPO-positive cells, which mostly represented neutrophils, were stained brown and clearly distinguished from other cells, such as lymphocytes, monocytes, and macrophages, which were stained pink. Following bacterial stimulation, the proportions of neutrophils were 27.49% and 38.05% in peripheral blood leukocytes and peritoneum, respectively, judging by the stained MPO. Kidney granulocytes contained abundant MPO-positive cells which were probably immature neutrophils with low expression of MPO. It is noteworthy that MPO-positive cells were detected in the tissue sections of kidney, spleen, and gut, with distribution profiles specific to each tissue. However, the cell morphology was not distinct in the stained tissue sections. These results indicate that the KI-PyY staining method is highly sensitive, applicable to different types of samples, and will be useful for the study of neutrophils in different compartments of fish.     

Vasopressin in tissue basophils of the dura mater of white rats

By an indirect immunohistochemical method with fluorescein-isothiocyanate (FITC) and horse radish peroxidase as markers (HRP) the presence of vasopressin was shown in cells of dura mater in white rats. Mast cells were identified after staining with methylene blue by the metachromatic granularity of the cytoplasm. It was shown that the number of cells found by means of FITC luminescence corresponds with their number found by means of methylene blue. The use of conjugate with HRP unveils a lesser number of vasopressin-containing cells.     

Azoindoxyl methods for the investigation of hydrolases. IV. Suitability of various diazonium salts (author's transl)]

Using fresh frozen, freeze-dried or cryostate sections from aldehyde fixed rat tissues 13 diazonium salts were tested as simultaneous coupling reagents for the localization of acid, neutral and alkaline hydrolases with azo indoxyl methods. Hexazotized new fuchsine and/or Fast blue B are the diazonium salts of choice for the demonstration of acid beta-galactosidase, neuraminidase, beta-N-acetylglucosaminidase, acid phosphatase, and non-specific esterase followed by hexazotized p-rosaniline. Fast blue VB, BB and RR and Fast violet B are recommended for the investigation of alkaline phosphatase and lactase, Fast garnet GBC for acid beta-galactosidase, glucosaminidase and lactase. Fast red B, RC, RL and TR and Fast black K can only be employed for lactase studies. The exact concentration of the coupling reagent depends on the activity of the enzyme and the organ imvestigated. On the average 0.01-0.02 ml unstable diazonium salt/ml and 0.3--1 microgram stable diazonium salt/ml are sufficient for the correct localization of these hydrolases. Freeze-dried cryostat sections yield the best results in the demonstration of lactase and alkaline phosphatase independent on the coupling reagent used. Sections from formaldehyde or glutaraldehyde fixed organs are superior for the localization of the other hydrolases; an exception is the investigation of acid beta-galactosidase and glucosaminidase with Fast garnet GBC. Then, excellent results are obtained also with freeze-dried material. Fresh frozen sections are suitable for the localization of lactase with hexazotized new fuchsine or p-rosaniline and of alkaline phosphatase with Fast blue VB and BB or violet B. The total activity of acid, neutral and alkaline hydrolases can be investigated using semipermeable membranes in combination with all unstable and stable diazonium salts of choice. Reliable osmification of the azoindoxyl dye is only possible if hexazotized p-rosaniline is employed for coupling; without further posttreatment all azoindoxyl dyes are extracted by ethanol, isopropanol or xylol. 7 incubation media are given for the demonstration of hydrolases with azoindoxyl methods at the level of light microscopy for routine studies and typical examples for the application of these methods are presented. A modified procedure is described for the freeze-drying of cryostat sections with the Edwards-Pearse tissue dryer EPD3.     

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, CD3, MHCII 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 the 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.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Cytochemical Technique for Demonstration of Lipids,Polysaccharides and Protein Bodies in Thick Resin Sections

An effective cytochemical technique for the simultaneous demonstration of lipids, polysaccharides and protein bodies in the same section from the tissue embedded in Epon 812 is described. Thick sections of peanut cotyledon are used for a typical sample according to the following procelures. Firstly, PAS reaction: (1) Oxidize sections in 0.5% periodic acid in 0.3% nitric acid for 10 min, (2) Wash in running water for 1–2 min and then pass through distilled water, (3) Stain in Schiff's reagent for 30 min, (4) Wash in sodium metabisulfite 3 times, 2 min for each time, (5) Wash in running water for 5 min and then pass through distilled water. Secondly, Sudan black B staining: (1) Rinse section in 70% ethanol for 1-2 min, (2) Stain in fresh 1% Sudan black B in 70% ethanol for 30–60 min at 40–60℃, (3)Rinse in 70% ethanol for 1 min and then in distilled water. Thirdly, Coomassie brilliant blue R staining: (1) Rinse sections in 7% acetic acid for 1–2 min, (2) Stain in I% Coomassie brilliant blue R in 7% acetic acid for 20 min at 60℃, (3) Differentiate in 0.1% acetic acid for I min, (4) Rinse in lunning water for 5 min and then pass through distilled water, (5) Dry at room temperature or in oven, 40℃. The dry sections mount in glycerin-gelatin. After the above three step staining, the three main compounds of the cell can be stained simultaneously. Starch grains and cellulose cell wall take cherry red colour, lipids appear in black, protein bodies are blue. The sealed slides can be kept permanently.