STAIN REACTIONS

012005 Cells lose Gram positive character after cessation 

012006 Cells appear barred when stained by Gram methods. 

012027 Individual cells stain unevenly when stained by 

012009 Cells are acid fast by Ziehl-Neelsen method. 

012010 Cells are alcohol fast by modified Ziehl-Neelsen 

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When Stacey began to study color and staining reactions, the main interest of others was in the application and modification of the tests, but very little was known about the underlying chemistry. His work opened up new lines of investigation. 

Histochemistry of the Gram-staining Reaction for Micro-organisms, H. Henry and M. Stacey, Nature, 151 (1943)671. 

These comprise a group of organisms which, like the Gram-positive and Gram-negative groups, have been named after a staining reaction. 

Kramer, I. R. H. McLean, J. W. (1952). Alterations in the staining reactions of dentine resulting from a constituent of a new self-polymerizing resin. 

In the year 1994, T. Takao et al. published a staining reaction to identify radicalscavenging activity [21]. Takao et al. used the compound l,l-diphenyl-2-picrylhydra-zyl (DPPH), which changes its color, in the presence of antioxidants such as ascorbic acid or rutin, from blue-purple to colorless or yellow. Forty mg of DPPH has to be solved in 20 ml of acetone or methanol. The reaction is complete immediately. 

Based on its nature (aqueous solutions, physiological conditions, well-investigated labeling, and staining reactions) and the historical transition from slab-gel electrophoresis to CE, the main targets are biological and bioequivalent samples such as proteins, peptides, polynucleotides, oligonucleotides, and carbohydrates. 

Reports of allergic sensitivity to MDA are confounded by mixed exposures to chemicals such as epoxy resins and isocyanates, which make it difficult to relate specific cause with effect. MDA does appear to cause an intense yellow staining reaction involving the skin (especially fingers and palms), nails, and occasionally hair in exposed workers. The staining should serve as a marker for potential systemic exposure. 

Olesen, 0. V., A simpUfied method for extracting phenytoin from serum, and a more sensitive staining reaction for quantitative determination by thin-layer chromatography. Acta Pharmacol. Toxicol. 25, 123-126 (1967). 

Histochemical demonstration of most of the O-acetylated sialic acids is possible because substituents on the side chain of Neu hinder periodate oxidation of this part of the molecule to an extent dependent on the number and position of the O-acetyl groups already mentioned. Correspondingly, removal of these ester groups by alkaline treatment (0.5% KOH in 70% ethanol91) may increase the staining reaction of a sialic acid. For example, the presence of O-acylated sialic acids has been demonstrated in colonic, epithelial mucin of man and various mammals (summarized in Ref. 91), in healthy and diseased, human small-intestine,188-190 in bovine submandibular gland,182 in mouse and rat erythrocyte-membranes,191 and in human lymphocytes.192... 

Detect peroxidase activity using DAB by mixing 10 mg DAB (one tablet), 400 pL of 1 % cobalt chloride, 20 mL PBS, and 20 pL 30% hydrogen peroxide. Pour onto the blot, allow to react, and then wash with water to block the staining reaction (see Note 13)... 

Lindholm, A.M., Reuter, M. and Gustafsson, M.K. (1998) The NADPH-diaphorase staining reaction in relation to the aminergic and peptidergic nervous system and the musculature of adult Diphyllobothrium dentriticum. Parasitology 11 7, 283-292. 

Detection Wash at least three times for at least 5 min with T-TBS or T-PBS. Prepare the detection solution shortly before use. Treat the membrane with the detection solution (see Materials) until staining of the spots is strong enough. Stop the staining reaction by washing very thoroughly with distilled water. 

Multi-step technique (3) This is an indirect/direct method combining unlabeled primary antibodies with directly-conjugated antibodies. The method starts with staining the unlabeled antibody/antibodies with the appropriate detection system, but without performing the final enzymatic staining reaction. The tissue is blocked with normal serum from the host of the first primary antibody before the second, directly-labeled primary antibody is added. The staining ends with the two enzymatic reactions being performed sequentially. 

One positive tissue control should be included for each set of tests. Ideally this control should contain a spectrum of weak to strongly positive reactivity. If such tissue is not available, another option is to select a weakly positive tissue, as this provides the best basis to evaluate whether a particular staining reaction is too weak or too strong. 

Add 2 x 105—1 x 106 cells/staining reaction to 5 mL FACS tubes or to V-bottom 96-well plates for FACSArray. 

Assay Control The assay control involves omission of the primary antibody (test article or negative control antibody) during the staining reaction and permits determination of staining by the secondary or tertiary antibodies or other components of the reaction process. This control slide may or may not be included based on the immunohistochemical method chosen. 

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