We adapted the peroxidase-labeled antibody method to localization of immunoglobulins and the secretory component (SC) in human intestinal mucosa at the light and electron microscopic levels. Modifications of the method included (1) the use of digitonin in the first wash step of tissues fixed in periodate-lysine-paraformaldehyde; (2) double coating of glass slides with gelatin and egg albumin before application of cyostat sections, and (3) inactivation of endogenous peroxidase by periodic acid treatment of cyostat sections. With these improvements, tissue morphology was well preserved; antigenicity of immunoglobulins and SC was retained; penetration of labeled antibodies into cellular compartments was complete; and confusion at the light microscopic level between peroxidase-containing cells and cells stained with peroxidase-labeled antibodies was avoided.
Potassium-osmium-cyanide complex (POCC) was substituted for osmium tetroxide procedures for the ultrastructural demonstration of cytochrome oxidase and peroxidase with diaminobenzidine (DAB). POCC reacted with DAB polymer resulting from the oxidation reaction producing electron opaque deposits. POCC produced fine homogeneous staining and more contrast than did the usual osmication method. This suggests a means of making the methods more sensitive. Careful controls are necessary since POCC alone stains smooth membrane elements, thereby possibly obscuring true reaction products in some cases.
Immunofluorescent studies using antibodies against Tamm-Horsfall urinary glycoprotein were performed in order to study the localization of glycoprotein in the human, rat and dog urinary system other than kidney and in the prostate gland. All extracts from ureter, urinary bladder, urethra and prostate gland showed a single distinct zone of precipitation with the rabbit anti-Tamm-Horsfall glycoprotein antibody in the Ouchterlony double diffusion test. Tamm-Horsfall glycoprotein was observed in both the mucous membrane and the muscular layer of the urinary system other than the kidney, and also in the prostate gland.
The newly improved cryo-sectioning technique for electron microscopy has proven to be very versatile for investigating the fine structure of the alveolar cell wall in the lung. This communication is concerned with a fundamental critical evaluation of the technique with particular emphasis on the osmiophilic properties of pulmonary ultrastructural details. The results indicate the following; 1) Embedding media like gelatin and bovine serum albumin are not absolutely necessary and the simple use of sucrose as well as the dry cutting method described by Tokuyasu are sufficient. 2) Regarding the application of buffer solution for fixatives, phosphate buffer showed a special property to the alveolar lining layer by the deposition of osmium grains, but not in cacodylate. 3) Uranyl acetate demonstrates a higher preservation of the phospholipid in lamellar inclusion bodies and alveolar surface layer when it is applied as a fixative after the double fixations. 4) Application of partial dehydration using graded ethanols with 60 and 90% gave a distinctive appearance in the membrane systems probably as a result of sweeping excess amount of osmium grains over the tissue proper. 5) The membrane was not disclosed as unit structure as usually observed in conventional electron micrographs in any ways of this technique applied here.
By application of Lineweaver-Burk's graphical method to the histochemical system, glycogen phosphorylase and UDP glucose-glycogen glycosyltransferase (glycogen synthetase) activities were studied in hair follicles taken from both the bald and hairy regions of the scalp. The first discernible appearance of the newly formed polysaccharide was recorded at the end point of each point of each enzyme reaction. Although there was no difference between terminal and vellus hair follicles in the distribution of phosphorylase and glycogen synthetase, the activities were higher in the former. In both types of follicles, the bulbar sac was the site of the highest activity of all enzymes except phosphorylase b, which showed its highest activity in the lower portion of the anagen external sheath. Phosphorylase a activity in the lower external sheath was considerably lower than that in the bulbar sac, but not as low as glycogen synthetase activity. From these data it was concluded that the accumulation of glycogen in the lower external sheath is due to a decrease in phosphorylase a activity relative to glycogen synthetase.
The proliferation of the smooth-surfaced endoplasmic reticulum (SER) and the cytochemical localization of glucose 6-phosphatase (G6Pase) activity after phenobarbital treatment were observed in postnatal mouse livers showing rapid proliferation of SER and in adult livers. Animals received one daily intraperitoneal injection of phenobarbital for 3 or 5 days in a dosage of 35mg per kg of body weight for the first 3 days after birth, 50mg per kg for 3 to 9 days after birth, and 50 or 100mg per kg for the adult. Administration of phenobarbital to growing animals did not change the histochemical distribution of G6Pase activity within the liver lobule in contrast with reduction observed in the centrolobular activity in adult animals after the same dose of phenobarbital. An extensive proliferation of SER was noticed in centrolobular hepatocytes from adult animals treated with 100mg per kg of phenobarbital, while no such proliferation occurred after treatment with 50mg per kg of phenobarbital. However, centrolobular hepatocytes of 5- to 10-day-old animals showed a marked proliferation of SER with 50mg per kg of phenobarbital. These observations suggest that there are some differences in the nature of the phenobarbital-induced proliferation of SER between hepatocytes during the postnatal period and fully differentiated hepatocytes of the adult.
The efficiency of isoperoxidase labeled antibodies was examined using the antigen coupled Sepharose 4B system as well as the tissue staining technique. Four isoenzymes including acidic and neutral fractions proved to be useful as marker enzymes for immune reaction. Some differences in the specific and non-specific reactivities of the isoenzyme-antibody conjugates were also demonstrated.