Variation In Staining Intensity Research Articles

Quantitative immunocytochemistry of tyrosine hydroxylase in rat brain. I. Development of a computer assisted method using the peroxidase-antiperoxidase technique

We sought to develop a quantitative immunocytochemical procedure using the peroxidase-antiperoxidase (PAP) technique to analyze amounts of the neurotransmitter biosynthetic enzyme tyrosine hydroxylase (TH) within the nucleus locus coeruleus (LC) of rat brain. Rats were perfused with 4% paraformaldehyde. The brains were embedded in paraffin, sectioned at 5 μm in the sagittal plane and immunocytochemically stained with antibodies to bovine adrenal TH. Staining intensity, measured by a TV image analysis system was reproducible within±5%. Reaction conditions required so that the intensity of the PAP reaction product was directly and linearly related to the amount of TH enzyme protein in tissue were obtained by reacting tissues with saturating concentrations (2.5 mM) of diaminobenzidine (DAB) substrate, a constant dilution of antibody, and incubation time adjusted so that the darkest elements in tissue were below saturation. Variations in staining intensity of serial sections through the LC were found to be insignificant when compared to variations in staining between animals. In order to increase the amount of immunoreactive TH in the LC, groups of rats were treated with reserpine. Immunocytochemical and biochemical analyses were performed in parallel groups of control and reserpine animals allowed to survive 1–3 days following a single injection (10 mg/kg s.c.) of reserpine. A close correlation was found to exist between the amount of TH enzyme protein determined biochemically and the density of staining for TH. The maximal increase in TH measured immunocytochemically was 2.2-fold which was about 80% of the maximal induction determined biochemically. We conclude that the PAP method can be used for quantitative immunocytochemistry of brain TH providing that optimal reaction conditions are established.

Immunofluorescence and Histochemical Methods for Neural M1 Pyruvate Kinase Localization

The distribution of pyruvate kinase (ATP pyruvate phosphotransferase, EC 2.7.1.40) in the nervous system has been studied by both immunofluorescence and a histochemical procedure using nitro blue tetrazolium. The localization in various parts of rat central nervous system in situ, cerebellar and cerebral cortex, was compared to that found in vitro in cultures of cerebellum, spinal ganglia, cerebral astrocytes, and skin fibroblasts. (1) Pyruvate kinase was found predominantly in the cytoplasm of neuronal cell bodies. (2) Large neurons were better visualized than small ones. (3) No glial localization was clearly demonstrated in situ, although this does not rule out the presence of some M1 pyruvate kinase. (4) Regions expected to be rich in nerve terminals, such as the cerebellar glomeruli or the cerebellar molecular layer, showed intense staining even when the cell bodies themselves were negative. This was expected, owing to the previous demonstration of the presence of M1 pyruvate kinase in nerve ending by subcellular fractionation methods. (5) The localization was similar in situ and in tissue culture, except that nerve processes were better seen in the latter and astrocytes were sometimes stained in vitro. (6) Variation in intensity of staining was observed in similar cell types in the same section or in the same culture. This could represent different metabolic or functional or maturational states.

Evaluation of the glyoxal-bis-(2-hydroxyanil)-method for staining of calcium in model gelatin films and pancreatic islets.

The nature of tissue calcium, detectable with glyoxal-bis-(2-hydroxyanil), (GBHA), was investigated using gelatin films as model. The results indicate that in the films the procedure detects only the calcium fraction which was ionized in the original gelatin solution. The GBHA staining intensity (absorbance) appeared to be linear with the amount of ionized calcium in the range from 0 to 2 micrograms/cm2. The method allows detection of amounts of ionized calcium as low as 0.15 micrograms/cm2 or 0.0015 pg/mu2. For the measurement of calcium in pancreatic tissue of fed rats, the tissue was subjected to freeze-substitution at -80 degree C in acetone containing 1% oxalic acid. Adjacent sections were stained with either GBHA or aldehyde-fuchsin (AF). Exocrine tissue hardly stained with GBHA whereas islet tissue stained intensely. For GBHA as well as for AF a variation in staining intensity (visual evaluation) between islets was observed. Islet GBHA- and AF-staining intensities did not correlate. The AF-staining intensity but not the GBHA-staining intensity decreased with increasing islet diameter. Also in pancreatic islet tissue the GBHA method appears to be very sensitive and reproducible and small differences in islet GBHA-staining intensity can be detected. The results indicate that between islets differences in ionized calcium content exist. These differences do not correlate with the degree of B-cell granulation.

Ultrastructure of the atrioventricular node of the big brown bat, <i>Eptesicus fucus</i>

This investigation describes the ultrastructure of the atrioventricular node of Eptesicus fuscus. Two conducting cells types (nodal and transitional) are indentified which differ in location, myofibrillar content, and types of intercellular junctions. Centrally located nodal cells display variable staining intensity and contain disorganized myofibrils which rarely form sarcomeres. Desmosomes and nexus-like junctions connect the nodal cells. Transitional cells, situated peripherally, exhibit distinct sarcomeres and are attached to the adjacent cells through desmosomes and underdeveloped intercalated discs. Longitudinal arrangement of the mitochondrial cristae is frequently seen in both cells types. In the connective tissue stroma, numerous capillaries (with micropinocytotic vesicles), axons, and possibly axonal terminals, some filled with vesicles, are observed. A large ganglionated nerve trunk is present on the nodal periphery. True nexuses and neuromuscular junctions are not observed. It is suggested that nodal cell types previously reported in different vertebrates under various names are merely simple variations of the two basic types of conducting cell--nodal and transitional. No interspecific differences are observed between these cells of the big brown bat compared with those in other vertebrates.

X-Ray and Proton-Induced Ultrastructural Changes in Dividing Chlamydomonas reinhardi

Early in the dark period of a 12-hr light, 12-hr dark synchronization regime cells of Chlamydomonas reinhardi are either in division or preparing for it, and are more sensitive to x and proton irradiation as expressed in terms of mortality and mechanisms of death exhibited by lethally injured cells. Irradiated and control cells were fixed for electron microscopic examination at intervals up to 5 days postexposure. X irradiation with 9000 R produced 69% mortality in 137c wild type cells and 71% mortality in the acetate-requiring strain ac-31. Ultrastructural responses observed in x-irradiated cells included nuclei with nonnuclear condensations and swollen nuclear envelopes, nuclear envelope rupture in cells in an advanced state of disorganization, multinucleate cells and changes in chloroplasts and mitochondria. Irradiation of dividing 137c cells with 1.5-MeV protons possessing a dE/dx of 200 MeV cm/sup 2//g produces 89% mortality following 9 krad and 98.3% following 18 krad. Proton-induced changes resembled those observed following x irradiation. Multiple nuclei per cell and variations in staining intensity of entire cells or parts of cells are documented. Two features absent in control cells were of special interest following both types of irradiation: (1) Cells were observed which remained joined in configurations closely resemblingmore » division profiles or division conformation. (2) Chromosomes and spindle fibers were present at a time not characteristic of the normal cell cycle.« less

LH Receptors and Leydig Cell Responsiveness to hCGin vitro

The in vitro responsiveness of isolated Leydig cells from 30-day-old rats to hCG was assessed in terms of androgen and cAMP production, under conditions where the quantity of LH/hCG receptors on the Leydig cell membranes was reduced to varying degrees. Reduction in the receptor population was achieved by a single, in vivo injection of 75 IU hCG 3 and 5 days prior to incubation. When receptor binding was reduced by approximately 30%, neither the sensitivity (dose of hCG needed to give a half maximum response) nor the magnitude of the Leydig cell response to hCG in terms of androgen production was affected. However, a reduction in [125I]-hLH receptor binding by 80% was associated with a decrease in the in vitro sensitivity of Leydig cells to hCG, resulting in a displacement of dose-response curve to the right. Maximal quantity of androgen produced was not reduced. In the case of cAMP production, the same reduction in LH binding was associated with a decrease in both sensitivity and magnitude of the maximal response. The number of LH/hCG receptors has to be reduced more than 30% in order to have significant effects on the steroidogenic response. Immunofluorescence studies of LH/hCG receptor in a normal Leydig cell population revealed a striking intercellular variation in intensity of peripheral membrane staining, indicating great differences in number of LH receptors on individual cells. Three days after injection of hCG (75 IU), this variation in staining intensity was even more pronounced. The injection caused a generalized decrease in staining intensity and reduced the number of cells with distinct membrane fluorescence by 60% despite the fact that the number of Leydig cells identified by morphology and by the presence of 3β hydroxy steroid dehydrogenase was not changed. The reduction in [125I]-hLH binding 3 days after the injection of hCG was not the result of endogenous occupancy (due to the injected hCG), but a result of disappearance of LH/hCG receptor from Leydig cell surface. The effect of hCG on membrane staining of Leydig cells indicates a general loss of LH/hCG receptors rather than a selective loss from a subpopulation of cells.

Immunocytochemical localization of epidermal growth factor in mouse submandibular gland.

The cellular and subcellular localization of epidermal growth factor in the submandibular glands of male and female adult mice was established by immunoperoxidase techniques. In light microscopic preparations epidermal growth factor was found exclusively in the granular convoluted tubules of the gland. The intensity of staining for epidermal growth factor varied from cell to cell, and some cells apparently were negative. The pattern of staining was similar in the glands of male and female mice; however, the granular convoluted tubules are androgen-responsive, and thus more extensive and composed of larger cells in males. In thin sections epidermal growth factor was most heavily concentrated in the secretion granules of the granular convoluted tubule cells. Within a given cell there was variation in intensity of staining of individual secretion granules, with some granules appearing minimally reactive or negative. The only other cell component with deposits of reaction product was the ribosomes.

Histological and histochemical observations on the eosinophilic vesicles found in the head eidermis of Blepsias cirrhosus (Pallas). A preliminary report.

1. This paper is a preliminary report on the eosinophilic vesicles found in the head epidermis of Blepsias cirrhosus.2. Such vesicles occurred also in the following three species: Cottus nozawae, Hemitripterus villosus and Myoxocephalus sp. Accordingly, the vesicles seemed to be not a morbid structure, but a wide-spread one among the fishes belonging to the family Cottidae.3. They were roundish oval or pear shaped, and of variable sizes, and their tips occasionally opened to external. They were generally prominent in the species supplied with few mucous cells.4. The contents of the vesicles looked light yellow or colorless, and were stained more easily with acid dyes, especially with eosin, than with the basic ones. They exhibited considerable variations in staining intensity and color tone according to whether they consisted of gross granules or fine ones.5. The chemistry and the function of these vesicles need further investigation.

The effect of estrogen on the histology of the uterine epithelium of the mouse: II. Changes of pas-reactive structures in the basement membrane and the glandular cell surface

The basement membrane in the uterine epithelium of spayed mice is thick and folded and shows an intense PAS reaction. After estrogen treatment and at estrus it follows a straight course, becomes thinner and is only weakly stained. In early pregnancy the folding and strong PAS reaction reappear. Electron microscopy shows that the dimensions of the basement membrane are unchanged. The variations in staining intensity are therefore due to different degrees of folding in a membrane with constant thickness. This folding depends chiefly on the state of the epithelial cells. A layer of intensely PAS-reactive material appears on the surface after estrogen treatment and at estrus. In the electron micrographs increase in length of the microvilli and deposition of dense strands of an amorphous substance between them are observed. This substance is assumed to represent secretion from the epithelium.

Automation in Staining of Cytologic Specimens

With continuous automatic staining procedures, it is now possible to stain hundreds of cytologic specimens per hour with a minimum of effort, and with reproducible optimum results. Two automatic and continuous polychrome staining procedures for cervical-vaginal specimens, and two automatic and continuous hematoxylin-eosin staining procedures for abrasion biopsies are presented. The continuous and automatic procedures reduce the number of solutions and the number of procedural steps needed for cytologic staining. They are simpler, less time-consuming, less costly and give fewer chances for error. They assure reproducible staining results even with less experienced technicians. They permit the technician to be occupied with other activities while the greater part of the staining is being done. They require only the part-time use of the Autotechnicon. They permit variation in staining intensity and color differentiation to suit individual preferences. Their use may be of critical importance in the developmen...