After aldehyde-fixation, treatment with phosphotungstic acid (PTA) in aqueous acidic medium was shown to produce an intense electron-opaque stain with minimal distortion of organelles. Mitochondrial matrix, cisternae of the endoplasmic reticulum, and the Z-band of muscle were densely stained, whereas membranes stood out in negative contrast. Staining of glycogen or lipid was not apparent. Under certain conditions the stain density reflected the concentration of protein based on the quantitative reaction of PTA with the positively charged groups, although the stoichiometry of the reaction between PTA and protein varied with the kind of protein. The staining conditions established should provide a base for the use of the method in quantitative electron microscopy, particularly on thin sections.

Animals↗

PubMed1969

Measurement of thickness within sections by quantitative electron microscopy.

To apply the method of quantitative electron microscopy to the measurement of mass in thin sections, the thickness of the section at or very near the structure to be studied must be known. Dowex anion exchange resin AG 1 x 2, stained with phosphotungstic acid (PTA) at pH 6.4, was used as a thickness standard which could be embedded and sectioned. The sectioned PTA-Dowex appeared uniformly stained and exhibited suitable electron opacity. The stoichiometry of the reaction between PTA and the Dowex resin was measured by three independent methods based on gravimetric, colorimetric, and nitrogen determinations whose results showed close agreement. From the PTA uptake, the density of the stained spheres was calculated. Mass of a defined area of PTA-Dowex was measured by quantitative electron microscopy, and from this mass and density, the volume and then the thickness were calculated. The values for thickness were compared to those obtained by interference microscopy on the embedding medium alone in the same sections.

Colorimetry↗

PubMed1969

Measurement of protein concentration by quantitative electron microscopy.

The method of quantitative electron microscopy was applied to the measurement of protein concentration in thin sections. The human erythrocyte was selected as a model because of its apparently uniform protein concentration. Phosphotungstic acid (PTA) in aqueous solution was used as a reversible stain for protein, and PTA-stained Dowex resin spheres were embedded along with the red cells as standards for measurement of section thickness. The mass of stain removed from a given area of sectioned red cell by buffer (pH 7.4) was measured by quantitative electron microscopy. From the stoichiometry of the reaction between PTA and red cell protein established in this study, the amount of protein present in the measured area was calculated. From this amount of protein and the measured thickness, the concentration of protein was calculated and expressed as g/100 ml, for comparison with the clinical laboratory value for hemoglobin. Groups of red cells from the same sample were measured on 3 different days and their mean values (g/100 ml +/- SD) were 29 +/- 3.9, 30 +/- 2.7, and 33 +/- 4.6, compared to the clinical laboratory value of 32.1 g/100 ml packed cells, after correction for volume change and protein loss during fixation.

Blood Proteins↗

PubMed1969

Quantitative histological distribution of serotonin in the rat stomach.

The quantitative histological distributions of serotonin in the body of the glandular stomach of the fed, fasted, and adrenocorticotrophin [ACTH]-injected rat were established. A tendency to greater serotonin concentrations in the epithelial cell region was found in both the fed and ACTH-treated (10 mg of ACTH in 1 ml of saline, intraperitoneally, 1 hr before killing) states. An elevated concentration observed in the submucosal zone of the fed and fasted animals was shifted to the chief cell zone following the ACTH treatment of the fed rats. This treatment was not given to the fasted animals. Relatively low concentrations were seen in the parietal and mucous neck cell region, in the chief cell zone except for the ACTH influence, and in the muscle. The higher concentration of serotonin in the submucosa may be related to increased innervation and numbers of mast cells, the greater concentration in the epithelial area to mast cell number, and the presence of material on the surface of the mucosa. The latter might also account for the greater concentration in the epithelial zone of the fed rats compared with the fasted rats. Clear differences are apparent in the distribution of serotonin reported in this communication and of histamine reported earlier.

Adrenocorticotropic Hormone↗

PubMed1969

Effect of vagotomy on the quantitative histological distribution of histamine and histidine decarboxylase in the rat stomach.

Determinations were made of the quantitative histological distribution of histamine and histidine decarboxylase in the body of the glandular stomach of fed and 14-hr fasted rats subjected to pyloroplasty and to pyloroplasty with vagotomy for comparison with untreated rats. Pyloroplasty on fasted rats had no significant effect on the concentration of histamine which was localized predominantly in the chief cell zone. However, the superimposed vagotomy was associated with some increase in the concentration. With the fed rats, the concentration was essentially unchanged from normal in either group of operated animals and it also was about the same as that in the normal fasted group. Histidine decarboxylase activity, which was also localized predominantly in the chief cell zone, underwent an increase in concentration following pyloroplasty of the fasted rats; no additional effect was observed with added vagotomy. The higher concentration of the enzyme activity in the fed rats was markedly further elevated in both groups of operated animals but these groups did not differ significantly from one another. An explanation of the elevation of histidine decarboxylase activity following pyloroplasty requires further investigation, but the lack of effect of the superimposed vagotomy on the enzyme activity would appear to rule out direct vagal control as the sole factor in determining the enzyme function at this site.

Animals↗

PubMed1968