amido-black and Carcinoma--Ehrlich-Tumor

Conditions are described by which cells and fresh frozen tissues, following fixation with ethanol-ether, and after staining with the amidoblack (AB)-TCA-staining method, show a modified dye/protein ratio of 0.9 moles AB/10(5) g protein compared to 8.5 moles AB/10(5) g protein ( Schauenstein et al. 1980) as in a previously used method. In contrast to the AB-TCA-method, which leads to extremly high and unmeasurable extinctions in tissue sections, staining with the modified AB-TCA- 23st -method with 10 microns tissue sections produces easily measurable extinction values. A correlation of the microspectrometrically determined mean total extinction values of different cell types and nuclei after staining with the tetrazonium method (N ohammer 1978; N ohammer and Desoye 1981) and on the other hand with the AB-TCA 23st -method has been found. The microspectrometrically determined extinctions after AB-TCA 23st -staining can be calculated; an extinction of 0.04248 corresponds to 1 pgm protein.

Topics: Amido Black; Animals; Azo Compounds; Carcinoma, Ehrlich Tumor; Liver; Mathematics; Proteins; Rats; Spectrophotometry; Staining and Labeling; Trichloroacetic Acid

In aqueous solution Amido Black B (ASB) forms stable and well-defined complexes with bovine serum albumin (RSA) at pH 5.5. The complexes can be separated by column chromatography. The formation of the complexes consist in a fast reaction during which, after 3 to 5 h approximately, 3 molecules of ASB have been bound per molecule RSA, and of a much slower reaction which, even after a laps of 24 h, is still far from approaching its final stage. With solid films of RSA, after denaturation with ethanol, fast reaction is found to approach its final stage after 10 min reaction time. With these model protein preparations, the molar extinction coefficient of the ASB-protein complexes can be determined: the soluble ASB-RSA complexes can be brought to complete dissociation at pH 12.3. After the additivity of the specific absorptions of both RSA and ASB had been proven, it was possible to determine the content of the solution of ASB and RSA, and therefrom the molar extinction coefficient of the ASB-RSA-complex at Ph 5.5: epsilon 620 = 110,000. ASB-stained ethanol-fixed RSA films show an epsilon 620 of approximately 96,000, if their thickness and specific weight are known. After incubation in watery or ethanolic/TCA solutions of ASB, also animal cells fixed with ether-ethanol show the ASB absorption band to be in the region of 600 nm after removal of the surplus of ASB by thorough washings. As already observed with the RSA films, the kinetics of the staining of the cells show the fast reaction reaching its final stage already after 15 to 20 min. When alcoholic solution of ASB is used, the extinctions are found to be twice or three times higher than those achieved by an aqueous one. After standardization of the staining procedures with both solvents the total extinctions of EATZ, YATZ, rat hepatocytes, chicken thymus and bursa cells were measured and plotted against the macroscopically determined protein content of the respective cells. Highly significant positive linear correlations resulted with staining both in watery and alcoholic solutions, respectively. From the slope of the straight lines, specific extinction coefficient of ASB stained cellular proteins could be calculated up to epsilon' 620 = 1.76 with watery ASB solution and epsilon' 620 = 3.83 with the alcoholic solvent. The soluble ASB-RSA complexes have an epsilon' 620 = 1.67 the ASB stained ethanol denaturated films of RSA an epsilon' 620 of within a range of 1.21 to 1.80.

Topics: Amido Black; Animals; Bursa of Fabricius; Carcinoma, Ehrlich Tumor; Histocytochemistry; Liver; Lymphocytes; Mice; Neoplasm Proteins; Protein Binding; Proteins; Rats; Sarcoma, Yoshida; Serum Albumin, Bovine; Thymus Gland