A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity.

We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using four histochemical techniques: ELF-97 phosphate, Gomori method, BCIP/NBT, and naphthol AS-MX phosphate coupled with Fast Blue BB (colored) and Fast Red TR (fluorescent) diazonium salts. Each method localized endogenous alkaline phosphatase to the same specific sample regions. However, we found that sections labeled using ELF-97 phosphate exhibited significantly better resolution than the other samples. The enzymatic product remained highly localized to the site of enzymatic activity, whereas signals generated using the other methods diffused. We found that the ELF-97 precipitate was more photostable than the Fast Red TR azo dye adduct. Using ELF-97 phosphate in cultured cells, we detected an intracellular activity that was only weakly labeled with the other methods, but co-localized with an antibody against alkaline phosphatase, suggesting that the ELF-97 phosphate provided greater sensitivity. Finally, we found that detecting endogenous alkaline phosphatase with ELF-97 phosphate was compatible with the use of antibodies and lectins. (J Histochem Cytochem 47:1443-1455, 1999)     

Light microscopic demonstration of myoid material in nuclei

Summary In the rabbit and bovine cornea the activity of alkaline phosphatase using histochemical as well as biochemical methods was investigated. Biochemically the enzyme activity was studied in separated corneal layers. In the histochemical investigation the best results were obtained in cryostat sections using the azocoupling method with naphthol AS-MX phosphate and Variamine Blue RT Salt. The enzyme activity was found not only in the epithelium and endothelium (as was described previously) but even in keratocytes. The mutual relation of activities in the epithelium and in keratocytes differed in both species. The overall activity found by histochemical methods is in good agreement with the biochemical determination of alkaline phosphatase (p-nitrophenyl phosphate as the substrate). Besides the histochemical approach shows an uneven distribution of alkaline phosphatase activity in individual cells which cannot be assessed by the biochemical determination.     

A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining and immunoenzymatic labeling.

We have developed a reliable and sensitive immunohistochemical staining technique which allows the simultaneous demonstration of two different antigens expressed in or on the same cell (referred to as mixed labeling), together with the evaluation of the general histopathological appearance of the tissue. The staining procedure combines a three-step (streptavidin-biotin) immunogold-silver staining (IGSS) with a three-step immunoenzymatic labeling. For this purpose, we investigated the compatibility of IGSS with various substrates of peroxidase or alkaline phosphatase (AP). Highly reliable and discernible mixed labeling was achieved only after initial labeling with IGSS followed by AP labeling using the substrates naphthol AS-MX phosphate/Fast Blue or naphthol AS-BI phosphate/New Fuchsin, respectively. To ensure utmost specificity, we applied FITC-conjugated mouse monoclonal antibodies and rabbit anti-FITC immunoglobulins visualized by AP-labeled immunoglobulins and the respective substrate in a final step. This novel approach provides an excellent means for demonstration of immunocompetent cells and unequivocal determination of the percentage of specific cell subsets in infiltrated tissue. The advantages of this method, as compared with double immunofluorescence or double immunoenzymatic labeling, were investigated and are discussed.     

Acute lymphoblastic leukemia hand-mirror cells with OKT 8 phenotype

Acute lymphoblastic leukemia with hand-mirror cells (ALL-HMC) was diagnosed in a 20-year old patient. Cytochemical investigations revealed a positive reaction for PAS and acid phosphatase. Lymphoid blast cells were studied with various monoclonal antibodies in order to determine their derivation and differentiation. The data obtained (positivity for Leu 9, OKT 11 and OKT 8) suggest that blast cells were of T lineage with OKT 8 phenotype. This report supports the phenotypic heterogeneity of ALL-HMC.     

Improved Immunohistochemical Visualization of Helper/Inducer T-Cells by the Simultaneous Application of two Noncrossblocking Monoclonal Antibodies

We have studied the intensity of staining of helper/inducer T-cells in lymph node and tonsillar tissue using two commercially available monoclonal antibodies (OKT4 and Leu3a) with the indirect immunoperoxidase method. Paracortical and mantle zone helper/inducer T-cells were easily visualized by both monoclonal antibodies, but T-cells in the follicular center, though stained by Leu3a, were hardly demonstrable by OKT4. Excellent staining was obtained in the indirect immunoperoxidase procedure by incubating the sections with a 1:1 mixture of the two monoclonal antibodies which gave bright staining of individual cells throughout the lymphoid tissue. Dilution of the primary antibodies by 1:200 did not affect the results. It is concluded that the simultaneous application of OKT4 and Leu3a as primary antibodies in the indirect immunoperoxidase procedure is the method of choice for the in situ demonstration of helper/inducer T-cells.     

Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures

Summary Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline phosphatase conjugated avidin. The lectin binding sites were visualized after incubation in substrate media containing: (1) 5-bromo-4-chloro indoxyl phosphate and Nitro Blue tetrazolium or copper sulphate; (2) naphthol AS-MX phosphate or naphthol AS-BI phosphate and various types of diazonium salts; (3) -naphthylphosphate and Fast Blue BB; (4) -glycerophosphate according to the method of Gomori. The results obtained with the alkaline phosphatase methods were compared with those seen with a streptavidin-horseradish peroxidase procedure. Several chromogen protocols for visualizing alkaline phosphatase activity showed differences in the ability to detect lectin binding sites. A sarcoplasmic reaction was evident for Con A, GS II, WGA, LCA, and PSA after incubation in the indoxyl phosphate medium. Sarcoplasmic reaction for GS II was also noticed after incubation with naphthol AS-MX Fast Blue BB and -glycerophosphate. The latter substrate also gave rise to a sarcoplasmic Con A reaction. With the indoxylphosphate tetrazolium salt method some muscle fibres showed a very strong intracellular reaction after incubation with Con A and GS II while the staining intensity was weak in other fibres. The same muscle fibres were stained with PAS. No sarcoplasmic reactions were observed with either naphthol phosphate media or with the diaminobenzidine peroxidase methods. Further, the staining of the muscle fibre periphery, connective tissue, and capillaries was intensified using the indoxyl method. The indoxylphosphate-tetrazolium salt method seems to be suitable for future investigations of lectin binding sites in muscle sections.     

Improved immunohistochemical visualization of helper/inducer T-cells by the simultaneous application of two noncrossblocking monoclonal antibodies

We have studied the intensity of staining of helper/inducer T-cells in lymph node and tonsillar tissue using two commercially available monoclonal antibodies (OKT4 and Leu3a) with the indirect immunoperoxidase method. Paracortical and mantle zone helper/inducer T-cells were easily visualized by both monoclonal antibodies, but T-cells in the follicular center, though stained by Leu3a, were hardly demonstrable by OKT4. Excellent staining was obtained in the indirect immunoperoxidase procedure by incubating the sections with a 1:1 mixture of the two monoclonal antibodies which gave bright staining of individual cells throughout the lymphoid tissue. Dilution of the primary antibodies by 1:200 did not affect the results. It is concluded that the simultaneous application of OKT4 and Leu3a as primary antibodies in the indirect immunoperoxidase procedure is the method of choice for the in situ demonstration of helper/inducer T-cells.     

Acid hydrolases in the suckling rat small intestine. II. On the importance of alkaline phosphatase inhibition in the histochemical localization of acid phosphatase activity.

Phosphatase activities against beta-glycerophosphate, I-naphthyl phosphate and naphthol AS-TR phosphate were investigated, at acid and aldaline pH levels, using unfixed and fixed cryostat sections of suckling rat jejunum. The use of 10 mm EDTA and 10 mm NaF as inhibitors indicated that alkaline phosphate is predominantly located in the microvillous region of the adsorptive cells, while acid phosphatase is located in small particles distributed between the brush borders and the nuclei of these cells. Alkaline phosphatase activity was found to interfere with the localization of acid phosphatase unless EDTA was included in the incubation medium. A modified Gomori medium, containing 10 mm EDTA and additional lead nitrate, is described. Latency experiemtns using this medium, with unfixed sections, indicated the lysosomal nature of particulate acid phosphatase. The discussion stresses the importance of including an aldaline phosphatase inhibitor in incubation media designed to localize extralysosomal acid phosphatase activity.     

Use of monoclonal antibodies to human lymphocytes to identify lymphocyte subsets in lymph nodes of the rhesus monkey and the dog

Using commercially available monoclonal antibodies that bind to human lymphocyte subsets, we examined lymph nodes from the rhesus monkey and the dog for their binding ability to these monoclonal antibodies. The avidin biotin-peroxidase immunostaining procedure was used, and the following antibodies were reactive in the rhesus monkey: Leu 4, Leu 3a, OKT4, Leu 2a, OKT8, T8, Leu 5b, T11, Leu 14, B1, and Leu 12. No immunoreactivity was observed in the dog lymph node except for moderate reactivity of OKT8. The following antibodies failed to react in both the rhesus monkey and the dog: OKT3, T1, T2, T1B, T4, T8A, Pan B, and T29/33. Kappa and lambda immunoglobulin light chains were positive in both the dog and monkey.     

A Quantitative Histochemical Study of Alkaline Phosphatase Activity in Isolated Rat Duodenal Epithelial Cells

A simple separation method enabling the quantification of alkaline phosphatase activity in unfixed, isolated, individual, duodenal epithelial cells has been presented. The activity of intestinal brush border-bound alkaline phosphatase has been demonstrated using naphthol AS-BI phosphate as a substrate and hexazotized New Fuchsin as a simultaneous coupling agent. The amount of final reaction product, as measured cytophotometrically, increases linearly with incubation time (up to 10 min) and with substrate concentration (up to 0.4 mM). Maximum enzyme activity was obtained at pH 8.9. Variation of the substrate concentration revealed the kinetic parameters for naphthol AS-BI phosphate as K_m = 0.17 ± 0.015 and V_max = 13.9 ± 1.38. The specificity of the enzyme reaction was confirmed by the complete inhibition of the enzyme activity in the presence of l-cysteine (10 mm) and 80% inhibition with L - phenylalanine (30 mM). Comparison of alkaline phosphatase activity in 8-m cryostat sections (beginning at the tip and proceeding to the cryptal part) along the villus axis, with the activity of individual cells obtained by successive separation, revealed similar values of the percentage quotient derived from the entire activities in these two different methods. This suggests that the presented separation procedure gives rise to isolation of the respective cells from the corresponding areas of the villus. Finally, the isolated cells can be used as a valuable tool for the quantitative analysis of alkaline phosphatase activity along the length of the villus.     

Immunophenotypic characterization of primary and secondary lymphoid follicles

The need for an immunophenotypical referential framework relative to lymphoid follicle has led us to apply a panel of monoclonal and polyclonal antibodies, by means of a sensitive immunostaining method. Lymphoid follicle is an immunophenotypically complex structure made up of three lymphoid populations (B, being its bulk, and a few T and NK cells), dendritic reticulum cells (DRCs) and Flemming's macrophages. Follicular B population is To 15 +, B1+, OKB 7 +, HLA-DR + and C3bR +. In secondary follicles there are differential characteristic reactivities for each topographic compartment: Mantle zone is positive for OKB 2 and surface IgM (sIgM) and IgD (sIgD); germinal center (GC) clear zone (with centrocytic predominance) for OKT 9, sIgM and weakly for OKB 2; and GC dark zone (with centroblastic predominance) only for OKT 9. In sections, OKT 10 allows one to see immunoblasts and plasma cells, the latter being with lymphoplasmacytoid cells the only intracytoplasmic immunoglobulin holders. 10% of GC lymphocytes are T cells, almost exclusively T-helper (Leu 3a +). Another 10% to 15% of lymphoid cells are Leu 7 (HNK-1) +. In histological sections, DRCs are specifically marked with R4/23 and Flemming's macrophages with anti-alpha1-antitrypsin and anti-alpha1-antichymotrypsin antibodies, both populations being negative to OKM 1 and OKM 5.     

Assessment of a method for immunochemical detection of antigen on nitrocellulose membranes

Immunoblotting techniques are widely used for detection of antigen immobilized on nitrocellulose membranes. There are many immunolabeling methods and staining methods available to disclose the presence of antigen in such techniques. Five common staining methods each for alkaline phosphatase and horseradish peroxidase were examined. The staining methods with the highest sensitivity and the lowest background were selected for studies comparing five immunological labeling methods using human IgG as a model antigen. Results were evaluated on the basis of the least amount of detectable antigen and background staining. The most sensitive dot-blot method was then tested for its applicability to Western blots. For both dot-blots and Western blots, the immunoalkaline phosphatase methods are more sensitive than the corresponding immunoperoxidase methods. The use of biotinylated secondary antibodies and an avidin-enzyme conjugate is recommended. Disclosure of alkaline phosphate is best achieved with naphthol AS phosphate as substrate and fast blue BB as chromogen. Peroxidase is best stained using H2O2 and diaminobenzidine (DAB). Potential endogenous enzyme activities are demonstrable by blotting methods but can be inhibited by including levamisole in the disclosure reaction medium for calf intestinal alkaline phosphatase indicators, or by incubation of blots with sodium azide and hydrogen peroxide before immunolabeling when using horseradish peroxidase indicators.     

A study of acid phosphatase in mouse kidney using naphthol AS/BI phosphate as a substrate

Summary A kinetic study of mouse kidney acid phosphatase has been performed using an application of the histochemical method ofBurstone (1958a, b). The suitability of the use of naphthol AS/BI phosphate as a substrate for biochemical assays of acid phosphatase has been ascertained. However, the rate of inhibition of the enzyme by sodium molybdate and sodium fluoride suggests that naphthol AS/BI phosphate may represent a substrate for an acid phosphatase different from-glycerophosphatase.     

Spontaneous human T-cell cytotoxicity against murine hybridomas expressing the OKT3 monoclonal antibody: comparison with natural killer cell activity

Human peripheral blood lymphocytes (PBL) exhibited spontaneous cytotoxicity against OKT3 monoclonal antibody (mAb)-expressing murine hybridoma cells (OKT3 hybridomas). In contrast, other murine hybridomas expressing OKT4, OKT8, anti-HLA DR, and anti-HLA A, B, and C mAb were not lysed. PBL showed much lower levels of cytotoxicity (3 folds) against OKT3 hybridomas as compared with NK activity against the K562 targets. Lymph node (LN) cells exhibited the inverse relationship of cytotoxicity levels. The addition of OKT3 mAb to the effector cells totally blocked both the binding and the lysis of OKT3 hybridoma targets, indicating that the CD3 antigen on the effector cells may be involved in recognition of the targets. The addition of concanavalin (Con A) also inhibited the cytotoxicity of OKT3 hybridomas. OKT4 mAb-expressing hybridomas became susceptible to lysis after chemical attachment of OKT3 mAb with CrCl3. The kinetics of lysis of OKT3 hybridomas resembled that of NK activity. Both cytotoxicities were detectable after 1 to 2 hr and reached plateau levels by 4 to 6 hr. Effector cells responsible for lysis of OKT3 hybridomas expressed T3, T8, and Leu 7 antigens, but lacked T4 and Leu 11b antigens, and were sensitive to the treatment with L-leucine methyl ester. These results indicate that T3+, T8+, Leu 7+ and T4-, and Leu 11- granular lymphocytes have a spontaneous cytotoxic activity against OKT3 hybridomas which is different from classic NK activity. These findings may provide a method for the assessment of T-cell cytotoxicity mediated presumably by in vivo generated cytotoxic T lymphocytes in blood and the other immune organs.     

Human autologous rosette-forming cells. I. Expression of cell surface antigens in relation to age and lymphoid organ distribution

Autologous rosette-forming cells (auto-RFC) were characterized with monoclonal antibodies to various cell surface antigens using a technique combining immunofluorescence and rosette formation. In peripheral blood, auto-RFC were T cells (Leu 1+/OKT3+) the majority being derived from the helper/inducer subset (Leu 3a+/OKT4+). A small proportion of the circulating auto-RFC were Leu 2a+/OKT8+ and virtually none of them bore T10, T6, and DR antigens or peanut agglutinin (PNA) receptors. In the elderly, the percentages of Leu 3a+ auto-RFC increased significantly along with the augmentation of the Leu 3a+ circulating pool. After Con A stimulation of peripheral blood lymphocytes the autorosette population was expanded and therefore their phenotype was again that of helper cells. In the thymus, high levels of autorosettes are found (30 to 50%). Simple or double labeling of the rosetting cells with various monoclonal antibodies permitted the confirmation of the existence of distinct thymocyte subpopulations and moreover to identify the location of the auto-RFC in the intrathymic differentiation scheme. Nearly 70% of the rosetting cells were derived from common thymocytes, those cells defined by the coexpression of T10, T6, T4, and T8 antigens whether or not they were also stained by OKT3 antibodies. The remaining auto-RFC were found with similar frequency among the T4+ and T8+ mature thymocytes. In the spleen low percentages of auto-RFC were found and the majority resided in the Leu 3a+/OKT4+ population, similarly to peripheral blood autorosettes. Taken together, these data suggest that the expression of autologous erythrocyte receptors is acquired in the thymus and is gradually lost during T-cell maturation.     

A highly sensitive assay method for human placental alkaline phosphatase involving a monoclonal antibody bound to a paper disk

A monoclonal antibody which is specific for human placental alkaline phosphatase and does not cross-react at all with intestinal alkaline phosphatase was prepared, and a procedure for the determination of placental alkaline phosphatase activity in serum was developed involving this monoclonal antibody bound to a paper disk. The minimum amount of placental alkaline phosphatase detectable by this method is 0.0025 King-Armstrong unit. Good correlation with the heat-treatment method was obtained. Therefore this proposed method can be used as a routine clinical test for the determination of serum placental alkaline phosphatase.     

A multiple-staining procedure for the detection of different DNA fragments on a single blot.

We have developed a method that implies the use of a particular type of substrate which can be used in combination with alkaline phosphatase in detecting nucleic acid on filters. The method allows the detection of several different nucleic acid sequences on a single filter. In consecutive steps, the target DNA molecules are hybridized with different digoxigenin-labeled DNA probes. After each hybridization step, digoxigenin is detected with an antibody-alkaline phosphatase conjugate. This enzyme is subsequently visualized by a color reaction using different 2-hydroxy-3-naphthoic acid anilide (naphthol AS) phosphates as substrates in combination with varying diazonium salts. The multiple-staining procedure is based on the fact that the probe DNA-antibody complex can be removed while the color precipitate remains stably bound at its place on the filter. This allows several repeated hybridizations with other digoxigenin-labeled probes followed by antibody detection and color reaction with other naphthol AS phosphate-diazonium salt combinations. Aside from the ability to simultaneously visualize different target DNAs on a single filter, this new method provides several important features that are more powerful than the conventional 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (BCIP-NBT) color reaction for alkaline phosphatase. The colors are more stable and brilliant than BCIP-NBT; their development is faster, the resolution of closely spaced bands is greater, and the background is much lower. The detection limit for alkaline phosphates is as good as with BCIP-NBT (0.1 pg of DNA). One major advantage is the simplicity of removing the colors by ethanol incubation. In this paper, the method is described using the example of Southern blotted DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Automated histochemical analysis of cell populations in the intact follicle-associated epithelium of the mouse Peyer's patch

Summary A new technique of quantitative histochemistry has been developed to study the cellular composition of the follicle-associated epithelium of the mouse Peyer's patch. This technique involves applying naphthol AS-BI phosphate to the surface of intact tissue where it is hydrolysed by alkaline phosphatase present in the luminal membrane of the epithelial cells. Naphthol AS-BI produced by this reaction is then coupled to Fast Red TR diazonium salt at the site of hydrolysis. M cells present in the epithelium contain little alkaline phosphatase activity and, therefore, remain white. Treatment with Alcian Blue is finally used to label goblet cells. Subsequent quantitative analysis of alkaline phosphatase-rich cells is carried out by scanning microdensitometry. Using this technique it is possible to detect two populations of alkaline phosphatase-containing cells in mice reared in a normal animal house environment.These results are discussed in relation to possible interactions taking place between enteric antigens and the gut-associated lymphoid tissue which could reduce the ability of follicle-associated enterocytes to express alkaline phosphatase.     

A Stain for Bone—Illustrating Apposition and Absorption in Two Colors

Sections of either mineralized or demineralized tissues are incubated for 30 min at 37 C in a succinate-tetrazolium salt medium for the demonstration of succinic de-hydrogenase, and subsequently in a naphthol AS-MX phosphate and Red Violet LB salt mixture for 20 min at 25 C for the demonstration of alkaline phosphatase. Sections may be either mounted in a water-soluble medium, or dehydrated and mounted in a synthetic resin. Osteoclasts stain brown to purple; sites of osteoblastic activity, red.     

Natural killer cells in the blood and bone marrow of the rhesus monkey

Natural killer (NK) cells in peripheral blood lymphocytes (PBL) and bone marrow (BM) cells of the rhesus monkey were detected by their functional activity against K562 cells. Animals could be grouped into "high" or "low" NK responders, a trait found to be consistent over a period of 2 years. NK active cells in PBL were in the nonadherent population, with the majority bearing Fc receptors and a further subdivision of these into CR+ (complement receptor) and CR- NK cells. Of 10 monoclonal antibodies directed against different epitopes of human lymphocytes, OKT11, OKT10, and Leu 11 showed reactivity with rhesus NK cells. Only OKT10 was reactive with the effector site of the cell, as shown by its capability to block NK function. Of the Leu 11 monoclonal antibodies (a, b, c), Leu 11c was nonreactive while Leu 11a and Leu 11b were shown by immunofluorescence to bind to 7 to 21% of PBL; Leu 11b was also cytotoxic to the NK cells. Leu 11b did not prevent binding of Leu 11a to PBL, suggesting reactivity of these antibodies with different epitopes. Percoll fractionation of PBL and BM revealed a greater enrichment of NK activity with BM; also, with PBL peak NK activity occurred in fractions 4 and 5 while this occurred in fraction 5 with BM. Although Percoll PBL fractions contained a higher percentage of Leu 11b cells, the NK activity of the BM fractions was proportionately greater. The majority of PBL cells with NK activity were FcR+ while significant activity could be attributed to FcR- cells of BM, in both the unseparated and Percoll fractions of each tissue. The data suggest NK active cells of BM may be distinct from those found in PBL.