Peroxidase-antiperoxidase Complex Technique Research Articles

Somatostatin-containing and other endocrine cells in the pancreas of the spectacled caiman.

Light-microscopic immunocytochemistry was used to localize 4 major pancreatic hormones in the pancreas of the spectacled caiman, Caiman fuscus. Somatostatin, insulin, glucagon and pancreatic polypeptide were localized by the peroxidase-antiperoxidase complex technique. A relatively large population of somatostatin-containing D cells was present. The D cells were nearly as numerous as the insulin-containing B cells and glucagon-containing A cells which were the most common cell types. All three cell types were commonly intermingled with one another in endocrine cell areas. Pancreatic polypeptide-reactive F cells were absent from some regions of the pancreas, but where present were related to other endocrine cell types. Functional properties of the pancreatic endocrine cells in this anatomical variant remain to be determined.

42]Rat calbindin D28K: Purification, quantitation, immunocytochemical localization, and comparative aspects

The purification, and biochemical and immunological characterization of vitamin D-dependent rat renal CaBP have only recently been reported. These biochemical and immunological studies have permitted a more detailed analysis of this calcium-binding protein; thus, providing insight into the role of the vitamin D endocrine system in kidney and brain function. The purification method results in the isolation of electrophoretically homogeneous rat renal CaBP. Silver stain after analytical polyacrylamide gel electrophoresis of the purified protein resulted in the migration of CaBP as a single band, suggesting the homogeneity of the preparation. The molecular weight of renal CaBP determined by gel filtration on a calibrated Sephadex G-100 column was found to be 28,000. The calcium-binding properties were determined by equilibrium dialysis performed at 4° for 18 hours on a reciprocating incubation shaker in polystyrene bottles that had been washed five times with deionized water. A number of methods are available for immunolocalization of CaBP at the light microscopic level. The two methods described in this chapter are the peroxidase-antiperoxidase complex technique and the colloidal gold with silver intensification method.

Conditions for the immunohistochemical demonstration of complement factor C3 in formaldehyde-fixed and paraffin-embedded renal tissues

Paraffin sections of formaldehyde-fixed renal biopsies were labeled for complement C3 by a polyclonal rabbit antibody to human complement C3, by the peroxidase-antiperoxidase complex (PAP) and the avidin-biotin peroxidase complex (ABC) techniques, respectively. All tissues had C3 deposits according to direct immunofluorescence on fresh frozen sections. Staining for muramidase was introduced as an intrinsic control for the degree of tissue proteolysis after the necessary trypsin digestion prior to the immunoenzyme labeling. The results indicated that even minute deposits of C3 could be detected in paraffin sections by the ABC method, which was more sensitive than the PAP technique; the ABC method allowed a maximal dilution of 1:2,400 of the primary antibody as compared to 1:800 for the PAP technique.

Immunocytochemical studies of gonadotropin storage in developing castration cells.

The prompt nature of the postcastration rise of LH and FSH in the male rat suggests that there may be changes in hormonal storage patterns in developing castration cells. To study this phenomenon, pituitaries from male rats 12 and 24 h after castration were stained with antibovine LHβ (1:10,000) and antihuman FSHβ (1:8000) and two immunocytochemical methods: the peroxidase-antiperoxidase complex technique and the avidin-biotin complex technique. The postcastration rise in serum gonadotropins was determined by RIA. Developing castration cells showed dilated profiles of rough endoplasmic reticulum which contained stain for LH or FSH, or were empty and vacuolated. They did not stain for ACTH. Three stereometric parameters were evaluated: 1) the percentage of LH or FSH gonadotropes in the cell population, 2) the volume density or concentration of LH or FSH gonadotropes in the pituitary cell mass, and 3) the average area fraction (a) of individual gonadotropes, measured with a calibrated grid in the light micr...

Immunocytochemical localization of a folicle stimulating hormone-like molecule in the testis.

A follicle-stimulating hormone (FSH)-like molecule was localized in normal adult rat testes as well as testosterone-treated hypophysectomized rat tests with an unlabeled antibody (anti-FSH), peroxidase-antiperoxidase complex technique. Anti-FSH bound specifically to ultrathin sections of acrosomes of spermatids and intranuclear bodies of early spermatids. Quantitation of staining intensity demonstrated that FSH, used as an absorbing antigen, would significantly reduce this binding. There was less anti-FSH binding to the acrosomes of spermatozoa in the body and tail of the epididymis as compared to the less mature germ cells located in the testis and head of the epididymis. The acrosomal and nuclear staining of spermatids taken from hypophysectomized animals was similar to staining observed in sham injected animals. Taken together, these results suggest that there is a molecule within the acrosome that is immunologically similar to FSH. Most importantly, these results emphasize the importance of conducting physiologic experiments in conjunction with immunocytochemical studies.

Ultrastructural immunocytochemical characterization of the thyrotroph in rat and human pituitaries.

The beta chain of thyroid stimulating hormone (TSH) was localized in normal rat and human pituitaries with the unlabeled antibody--peroxidase-antiperoxidase complex technique and antisera to rat or bovine TSH beta. Araldite embedded tissues, immunoreactivity of TSH was best preserved after fixation with 1% glutaraldehyde or picric acid formaldehyde. In the TSH cells, the immunocytochemical stain was located on granules. However, there was a variation in staining of individual granules, and among the population of granules. Rat TSH cells were ovoid or angular to stellate, and contained granules ranging in size from 60-175 nm. The human TSH cell was polyhedral, and contained scattered granules of 150-300 nm diameter. In both species, granule distribution was either in 1-2 rows at the periphery, or scattered throughout the cytoplasm with some concentration in cytoplasmic processes. TSH cells in female rats in estrous contained more granules than those of other stages. TSH cells were distinguished from adrenocorticotropic hormone cells and luteinizing hormone cells on the basis of granule size and distribution, and cell shape.

Characterization by different techniques of adrenocorticotropin and gonadotropin producing cells in lerot pituitary (Eliomys quercinus)

Adrenocorticotropin and gonadotropin producing cells were localized in the adenohypothysis of normal Lerots by using anti-beta 1-24 ACTH, anti-LH, anti-LH beta, anti-PMSG antisera. In order to study their fine structure two techniques were employed: a superimposition technique which consists of detailed comparisons between the same cells in light, fluorescence and electron microscopic preparations and an immunocytochemical technique on ultra-thin sections using the peroxidase anti-peroxidase complex technique. The superimposistion technique allows an excellent description of cell ultrastructure of individually identified cells of each type. With this method we were able to describe the corticotropin secreting cells as lucent cells with electron dense granules ranging in size from 2500 to 3500 A. The gonodotropin secreting cells are darker and their granules are about 2000 A in diameter.

The Pituitary Response to Stress: An Immunocytochemical - Cytophysiologic Study

Physiologic studies have shown that there is a peptide in mammalian neurointermediate lobes with immunologic and biologic properties similar to adrenocorticotropin (ACTH). Immunocytochemists have localized the peptide in the secretory granules of the intermediate lobe parenchymal cells and in rostral zone anterior lobe ACTH cells. [Moriarty, G. C. and Halmi, N. S., Z. Zellforsch 132:1 (1972)].In an attempt to determine the physiologic significance of this peptide, we rapidly removed the pituitaries from intact and adrenalectomized rats which were stressed by ether (2 minutes) or a buzzer (30 minutes). The neurointermediate lobes were separated from the anterior lobes and prepared for electron microscopy and immunocytochemistry (with the unlabeled antibody peroxidase-antiperoxidase complex technique) and ACTH bioassay (with the use of dispersed adrenal cells according to Sayers et al.) and radioimmunoassay.