Naphthol AS phosphates

A section of the dorsal part is shown (p = parenchyma t = tegument). Immunolocalization was performed with an antibody against Schistosoma mansoni Hsp70 (Moser ef a/., 1990). Detection was performed using an alkaline-phosphatase conjugated secondary antibody, naphthol-AS-phosphate and Fast Red TR (Finken ef a/., 1994). 

APase has also been used for the probes to localize antigens in cells (Mason and Sammons, 1978), in the form of immune complexes of this enzyme (APAAP Section 11.3.2). Naphthol AS phosphate in combination with Fast Red TR gives an intense red reaction product which counterstains well with hematoxylin, whereas in combination with Fast Blue BBN it produces a blue color. 

Enzymatic revelation. First POase is revealed with DAB (brown color) and then APase with naphthol AS phosphate plus Fast Blue BBN, according to Section 17.3.3.2. 

A stock solution (10 mg/ml) of naphthol AS-MX, or naphthol AS phosphate, or naphthol AS-BI phosphoric acid sodium salt (Sigma) in DMF is prepared and diluted 50 times with 100 mM Tris-HCl buffer, pH 8.2. The solution is stable at 4°C for several weeks (in their original work. Mason and Sammons used naphthol AS phosphate and a buffer of pH 9.0). The substrate solution is prepared by dissolving Fast Blue BBN or Fast Red TR (1 mg/ml) in the naphthol stock solution. 

Fast Red gives the most intense (red) reaction product which contrasts well with hematoxylin. For double immunostaining (in combination with POase/DAB) Fast Blue is preferred due to its contrast with oxidized DAB. Naphthol AS-MX (Stage and Avra-meas, 1976) gives comparable results to naphthol AS phosphate. 

Fig. 7.7. Naphthol AS phosphate/diazonium salt colorimetric detection of APase is a very promising and cheap alternative to BCIP/NBT. Moreover, these substrates can be used to detect different probes on the same blot.

Developer Naphthol AS-BI phosphate (Sigma N4875) 2 mg, N,N-dimeth-ylformamid 0.2 mL, veronal-acetate buffer 9.8 mL, pH 9.2, levamisol 1 M... 

Develop with (naphthol AS-BI phosphate/Fast Red, fresh made) for 20 min in the dark (in tinfoil-sealed humidity chambers). For preparation of developer, see Subheading 5.1.1.1, item 4. 

Alkaline phosphatase acts on many substrates as well, each precipitating as a different color. For example, a combination of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) results in a permanent blue precipitate at the site of alkaline phosphatase localization. There are other compounds that can also be tried, such as Fast Red TR/Naphthol AS-MX (Sigma, St. Louis, MO), which precipitates as a red color. 

Develop the alkaline phosphatase with fast red dissolve 5 mg sodium naphthol AS BI phosphate in dimethylformamide (few drops) and add to 5 mg fast red TR salt in 10 ml veronal acetate buffer (pH = 9.2), incubate the slides for 1 h Wash with tap water Counterstain as desired... 

Additional substrates include naphthol AS-BI phosphate, naphthol AS-TR phosphate and 5-bromo-4-chloro-3-indoxyl phosphate (BCIP). Other possible chromogens include Fast Red LB, Fast Garnet GBC, Nitro Blue Tetrazolium (NBT) and lodonitrotetrazolium Violet (INT). 

Dissolve 2 mg naphthol AS-MX phosphate, free acid (Sigma N 4875) in 0.2 mL N,N-dimethylformamide in a glass tube. 

Substrate solution Add 10 mg of naphthol AS-MX phosphate (sodium salt) to a solution containing 30 mg of Fast Red dissolved in 100 mL of... 

Wash filters in 50 mM Tris-HCI (pH 8.2) and air-dry. Best results are obtained with naphthol AS-E phosphate and Fast Violet B but the others are very useful in multicolor detection procedures. 

Fast Blue chromogen-substrate solution Fast Blue BB Salt (Sigma) Naphthol AS-MX-phosphate (Sigma) N,N-dimethylformamide 0.1 M Tris-HCl buffer, pH9.0 and 1 M Levamisole (see Note 9) (see Subheading 3.3.5). 

To count multinucleated cells, cultured cells were stained as described below. After cells were cultured, adherent cells were fixed with 10% formaldehyde in phosphate-buffered saline (—) for 20 min. After cells were treated with 95% ethanol for 1 min, the well surface was dried and treated with tartrate-resistant acid phosphatase (TRAP)-staining solution [0.1 M sodium acetate buffer (pH 5.0) containing 50 mM sodium tartrate, 0.1 mg/ ml naphthol AS-MX phosphate (Sigma Chemical Co., St. Louis, USA), and 1 mg/ml fast red violet LB salt (Sigma Chemical Co.)] for 30 min. TRAP-(-l-) multinucleated cells were then counted under a microscope. 

Naphthol-AS-MX phosphate, disodium salt (Sigma Chemical Co.)... 

Using the Smith-Fishman technique which is based on the hydrolysis of naphthol AS-BI phosphate and on the visualization of naphthol AS-BI coupled to diazotized p-acetoxymercurianiline rather than on products of hydrolysis of /3-glycerophosphate, Sasaki and Fishman (1972) observed reaction product in the basal infolding membranes of mouse kidney cortex epithelial cells (Fig. 11). These structures would easily form vesicles during homogenization and should contribute to the microsome organelle population. This picture does make it possible to understand the biochemical findings now to be described. 

Immediately before use. dissolve 20 mg of naphthol AS-MX phosphate salt in 1 ml of N,N-diinethylformamide in a glass container. Add 20 ml of Tris buffer gradually with mixing, then add 12 mg of levamisole and 50 mg of Fast Red HI salt, mix well. 

CL reaction can be catalyzed by enzymes other than HRP (e.g., microperoxidase and catalase) and by other substances [hemoglobin, cytochrome c, Fe(III), and other metal complexes]. The presence of suitable molecules such as phenols (p-iodophenol), naphthols (l-bromo-2-naphthol), or amines (p-anisidine) increases the light production deriving from the HRP-catalyzed oxidation of luminol and produces glow-type kinetics [6, 7], The use of other enzymes, such as glucose-6-phosphate dehydrogenase [38-41], P-galactosidase [42], and xanthine oxidase [43-46], as CL labels has been reported. 

Develop the chromogeuic reaction of the second sequence using AEC/H2O2 development for IPO or naphthol phosphate/Fast Blue, Fast Red, or New Fuchsin for lAP development as detailed in Subheading 3.3. (see Note 15 for triple labeling). 

An interesting variation of the latter technique finds application in enzyme chemistry. In this procedure a tissue section is exposed to a relatively colorless derivative of j8-naphthol, such as sodium j8-naphthyl acid phosphate. A phos-photase enzyme reacts with this reagent (often called an enzyme substrate ), leaving free j8-naphthol behind. Subsequent treatment with a solution of a diazonium salt produces highly colored spots in the tissue section. Thus not only can the presence of phosphotase enzyme be demonstrated, but also the location of the enzyme in the tissue can be determined. The intensity and chroma of the color produced and the solubility of the azo dye in the cell materials can be varied by judicious selection of the reagents. 

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