Differentiation of Cells in the Hypophysis and in Pancreatic Islets

Deparaffinized, 3-5μ, sections are brought to water, oxidized 3.5 min in an equal-parts mixture of 0.3% H₂SO₄ and 0.3% KMnO₄, and decolorized with 4% K₂S₂O₅. Nuclei are stained with Gomori's (1939) chromium-hematoxylin, and cell granules with Cason's (1950) mixture. The eosinophilic cells of the hypophysis and the alpha cells of pancreatic islets (of Langerhans) stain carmine red; basophilic and beta cells stain dark blue. Heidenhain's _susa is the most suitable fixative for hypophysis, Bouin's fluid for pancreas; but a satisfactory result is obtainable after formalin-sublimate or plain formalin. Besides studying the ratio of the cell types in the hypophysis or in pancreatic islets, it is possible to estimate the granule content of the cells. The method works on human autopsy material provided fixation of hypophysis occurs within 24 hr, and. pancreas, 12 hr post mortem, and it is suitable also for quite fresh organs.     

A Staining Method for the Anterior Hypophysis of the Rat

A staining procedure for the anterior hypophysis of the rat, differentiating between eosinophilic granules, basophilic granules and mitochondria, has been divised. Small pieces of hypophyseal tissue are fixed in Champy's fluid. Following fixation the tissue is either chromated or osmicated. After being embedded in 60-62° paraffin, the tissue is cut serially at 2 and 3 μ. The sections are stained with 7% Altmann's acid fuchsin by heating on a laboratory hot plate, followed by 30 seconds in a 2% solution of Orange G made up in 1% phosphomolybdic acid. They are then treated for 10 seconds in a .01% solution of potassium carbonate, and stained for 10-30 minutes in Goodpasture's acid polychrome methylene blue. The mitochondria stain brilliant fuchsia, the eosinophilic granules orange-red, and the basophilic granules deep blue.     

Methods of Staining Plant Tissues for Differentiating the Natural Plant Oils from Petroleum Spray Oils

Petroleum, spray oils in sections of plant tissue have been distinguished from the plant oils by staining the fresh sections in the following dye solution: To a saturated aqueous solution of Nile blue sulfate, 0.5% sulfuric acid is added and the mixture is boiled under a reflux condenser for 4 or 5 hours. It should be as nearly alkaline as possible without a change of color. A solution of 50% alcohol and 50% acetone is then saturated with oil red O. One part of the Nile blue sulfate solution is then added to two parts of the oil red O solution. Allow to settle over night and filter. Stain several hours. Rinse in water and mount in glycerin jelly. A short discussion of the merits of this method and the differentiation of the spray oils by means of indophenol blue are also given.     

Differentiation of Cells in the Hypophysis and in Pancreatic Islets

Deparaffinized, 3-5μ, sections are brought to water, oxidized 3.5 min in an equal-parts mixture of 0.3% H₂SO₄ and 0.3% KMnO₄, and decolorized with 4% K₂S₂O₅. Nuclei are stained with Gomori's (1939) chromium-hematoxylin, and cell granules with Cason's (1950) mixture. The eosinophilic cells of the hypophysis and the alpha cells of pancreatic islets (of Langerhans) stain carmine red; basophilic and beta cells stain dark blue. Heidenhain's _susa is the most suitable fixative for hypophysis, Bouin's fluid for pancreas; but a satisfactory result is obtainable after formalin-sublimate or plain formalin. Besides studying the ratio of the cell types in the hypophysis or in pancreatic islets, it is possible to estimate the granule content of the cells. The method works on human autopsy material provided fixation of hypophysis occurs within 24 hr, and. pancreas, 12 hr post mortem, and it is suitable also for quite fresh organs.     

Impregnation of Oligodendroglia in Nervous Tissue Kept in Formalin for Many Years

A method for impregnating oligodendroglia in nervous tissue (monkey) fixed and preserved in formalin for many years is described. This tissue is reconditioned by placing 12 to 30μ frozen sections of it in concentrated ammonia (sp. gr. 0.90) and by washing them slowly for 24 hours with a 1 mm. stream of water. The fluid is then poured off the sections; the jar is refilled with concentrated ammonia; and washing is repeated for another 24 hours. The sections are then plunged into concentrated ammonia for 7 minutes.

After treatment in ammonia, the sections are incubated for one hour at 38^oC. in Globus' 5% hydrobromic acid solution. They are washed again, in distilled water, and then impregnated in a “medium” strength ammoniacal silver carbonate solution (5 ml. of 10% AgNO₃ added to 15 ml. of 5% Na₂CO₃. The precipitate is dissolved in concentrated ammonia and diluted to SO ml. with distilled water). Impregnation is followed by reduction in 1% formalin without agitation; fixation in 5% Na₂S₂O₃; dehydration, and mounting in clarite.

Typical oligodendroglia (Fig. 1) were made visible by use of the method outlined in this paper.     

A method for obtaining histological sections from tissue previously studied by scanning electron microscopy

An accelerated method of paraffin embedding of tissue specimens previously examined with scanning electron microscopy is proposed aimed to obtain sections for routine histological examination. The tissue is passed through acetone, absolute alcohol, alcoholic-oil celloidin solution, chloroform to be eventually mounted into paraffin. The method allows obtaining good quality sections within 24 hours.     

Ontogenesis of the α-MSH, β-MSH and ACTH cells in the foetal hypophysis of the rat. Correlation with the growth of the adrenals and adrenocortical activity

Pituitary sections from 15 to 21 day-old rat foetuses have been studied with the immunofluorescence technique, using antibodies anti alpha-MSH, anti beta-MSH and anti beta (1-24) ACTH. The first ACTH cells appear on day 17 of pregnancy in the pars distalis of the hypophysis and only on day 18 in the pars intermedia. beta-msh cells have been observed on day 16 in the pars anterior and on day 17 in the pars intermedia, while alpha-MSH cells appear only on day 18 and exclusively in the pars intermedia. The cytodifferentiation of the beta-MSH and ACTH cells occurs in the pars intermedia with about a 24 hours delay in comparison to that of the pars distalis. The first revealed cells are always located in the posterior half of the pituitary gland. The corticostimulating activity of the hypophysis has been tested with the fluorescence intensity of the corticotrophs, the adrenal weight, the adrenal content in corticosterone and the plasma corticosterone level. The fluorescence of the corticotrophs increases on day 18, shows a maximum on day 19 and decreases until term. The adrenal weight rises regularly between day 16 to day 20, thereafer the increase subsides. Adrenal and plasma corticosterone concentrations reach a peak on day 19 of pregnancy. These data suggest that hypophyseal corticostimulating activity is very high between days 18 and 19 and decreases between days 19 and 21.     

A Protargol Method for Staining Nerve Fibers in Frozen or Celloidin Sections

Extensive experimentation with protargol staining of neurons in celloidin and frozen sections of organs has resulted in the following technic: Fix tissue in 10% aqueous formalin. Cut celloidin sections IS to 25 μ, frozen sections 25 to 40 μ. Place sections for 24 hours in 50% alcohol to which 1% by volume of NH₄OH has been added. Transfer the sections directly into a 1% aqueous solution of protargol, containing 0.2 to 0.3 g. of electrolytic copper foil which has been coated with a 0.5% solution of celloidin, and allow to stand for 6 to 8 hours at 37° C. Caution: In this and the succeeding step the sections must not be allowed to come in contact with the copper. From aqueous protargol, place the sections for 24 to 48 hours at 37° C. directly into a pyridinated solution of alcoholic protargol (1.0% aqueous solution protargol, 50 ml.; 95% alcohol, 50 ml.; pyridine, 0.5 to 2.0 ml.), containing 0.2 to 0.3 g. of coated copper. Rinse briefly in 50% alcohol and reduce 10 min. in an alkaline hydroquinone reducer (H₃BO₃, 1.4 g.; Na₂SO₃, anhydrous, 2.0 g.; hydroquinone, 0.3 g.; distilled water, 85 cc; acetone, 15 ml.). Wash thoroly in water and tone for 10 min. in 0.2% aqueous gold chloride, acidified with acetic acid. Wash in distilled water and reduce for 1 to 3 min. in 2% aqueous oxalic acid. Quickly rinse in distilled water and treat the sections 3 to 5 min. with 5% aqueous Na₂S₂O₃+5H₂O. Wash in water and stain overnight in Einarson's gallocyanin. Wash thoroly in water and place in 5% aqueous phosphotungstic acid for 30 min. From phosphotungstic acid transfer directly to a dilution (stock solution, 20 ml.; distilled water, 30 ml.) of the following stock staining solution: anilin blue, 0.01 g.; fast green FCF, 0.5 g.; orange G, 2.0 g.; distilled water, 92.0 ml.; glacial acetic acid, 8 ml.) and stain for 1 hour. Differentiate with 70% and 95% alcohol; pass the sections thru butyl alcohol and cedar oil; mount.     

Sugar uptake by the grape berry: A note on the absorption pathway

Summary ³H-glucose was fed to excised Sultana grape berries via their pedicels for up to 5 hours. Autoradigraphy showed that the label was distributed throughout the fruit within 1 hour. Microautoradiography of tissue sections taken at a number of points showed that within the pedicel the walls of cortical cells had become heavily labelled, suggesting that the cortical cell walls offered a diffusion pathway for the solutes entering the vascular system from the external aqueous solution. Transport along the pedicel was confined to the central vascular tissue with little radioactivity occurring in the cortical cells. Within the pericarp, the vascular bundles and walls of nearby parenchyma cells had become heavily labelled, indicating that the labelled solute was present within the vicinity of cell walls. The general pattern of ³H-glucose accumulation by excised berries was similar to the deposition pattern of ²⁴C-labelled photosynthate within attached fruit.     

Histological Methods for the Demonstration of Gold in Tissues

Two methods for the demonstration of gold in tissues are described. The tissue is fixed in neutral formalin, embedded in paraffin, sectioned and run down to water. In the SnCl₂ method, modified from that of Christeller, it is then incubated for 24 hours at 56° C. in a mixture of ten parts of 5% SnCl₂·2H₂O and one part of concentrated HCl. The interpretation of the results obtained by this method is frequently difficult because of the presence of accessory precipitates and the presence of the normal pigments of the tissue. This has led to the development of a new method, in which the sections are incubated for periods varying from 24 hours to 6 days at 37° C. in 3% H₂O₂. The gold is reduced to the metallic state, the interfering tissue pigments are bleached, and, since no metallic ions have been added, accessory precipitates do not occur. After both methods, the sections are washed thoroughly, run up, and mounted in damar.     

Demonstration of dehydrogenase activities in paraffin sections.

The possibility of demonstrating the activity of respiratory enzymes in paraffin sections was studied. Unfixed pieces of nervous tissue were incubated at 4 degrees C, 20 degrees C, 37 degrees C and 56 degrees C for various periods ranging from 1 to 24 hours. After dehydration, the tissue pieces were mounted in paraffin. The paraffin sections obtained there of were then tested with respect to the range of penetration of the substrate into the incubated tissue samples (as judged from the resulting histoenzymic reaction), and for the distinctness with which the localization of the histochemic reaction could be assessed. From the results it may be concluded that it is possible, under well defined conditions, to demonstrate the activity of dehydrogenases in paraffin sections. The resulting morphological pictures permit a much better localization of the histoenzymic reaction products than those obtained from cryostat sections. Optimal results are obtained when tissue fragments, about 1 mm in diameter are incubated for 24 hours at 4 degrees C.     

Tissue changes in the rabbit periodontal ligament during orthodontic tooth movement

In this (semi) quantitative animal study the reaction of the periodontal ligament (PDL) to experimental tooth movement is described. To this end, rabbit first incisors were moved sideways with helical torsion springs for periods varying from 3-24 hours. The initial force of the springs was 50 gf. The histomorphology of the PDL was studied in 5 microns thick plastic sections. Comparison with control animals and animals wearing passive springs showed that tooth movement leads to an increased trauma in the PDL within only a few hours. This trauma is characterized by hyalinization, tears and ruptures in the fibres and blood vessels, and by the presence of extravascular erythrocytes and pyknosis. Tissue damage significantly increased with time. After 24 hours of tooth movement, the PDL fibers are compressed or stretched in 68% of the sections and the blood vessels in the PDL are compressed or stretched in 62% of the sections. Even in the controls, more than 15% of the sections displayed slightly stretched or compressed fibers, and about 10% showed slightly compressed or stretched blood vessels. This indicates that some damage is regularly present in a normally functioning PDL. Increases in the percentage of sections with blood vessel compression are found in all groups wearing passive springs, especially after 6 hours. A high concordancy in compression and tension patterns of blood vessels and fibers is present in 83% of the sections. Pyknotic cells are practically confined to areas with compressed PDL fibers in rabbits wearing active springs. Extravascular erythrocytes were found in sections with all types of fiber patterns. A significant majority of extravascular erythrocytes, however, was found in areas with compressed fibers.     

Uptake of immunoglobulin and albumin by granulated metrial gland cells in vitro

Single cell suspensions of metrial gland tissue from rats at Day 14 of pregnancy were prepared for maintenance in vitro. During the first 2 days of culture IgG was detected in glycoprotein granule-containing granulated metrial gland (GMG) cells. Albumin was also detected in GMG cells at the same stages. The IgG and albumin were not detected during the next 4 days in culture. When metrial gland cells, maintained in vitro for 5 days, were incubated with rat serum for a further 24 h, IgG and albumin were detected in GMG cells. When similar cultures were incubated for 24 h with purified rat IgG or purified rat albumin, GMG cells were positive for IgG and albumin respectively. Albumin was not detected in GMG cells in wax sections of metrial gland tissue, although IgG has previously been demonstrated. The uptake of serum proteins by GMG cells in vitro has been clearly shown but the difference in IgG and albumin content of these cells in paraffin-wax sections indicates that the means by which IgG accumulates intracellularly may be different in vitro and in vivo.     

Quantitative aspects of growth hormone cell maturation in the normal and little mutant mouse

Growth hormone (GH) cells were analyzed by means of ultrastructural morphometry in the pars distalis of pituitary glands from male adult and immature normal (C57BL) and homozygous little (lit/lit) mutant mice. Thin sections were exposed to anti-GH serum and processed immunocytochemically with the colloidal-gold technique. In the pars distalis of adult lit/lit mice, the mean volume density of GH cells/total tissue was 24% of the normal value, granules/GH cells was 58% of normal, and granules/total tissue was only 12% of normal. Deficits in all of these parameters likewise occurred in immature glands, though to a lesser extent than in the adults. The results indicate that the GH deficiency in this mutant reflects quantitative deficits in both the secretory granule content of GH cells, as well as the GH cell content of the gland, with the latter being the more severely affected.     

A Golgi-Electron Microscope Method for Insect Nervous Tissue

Golgi's light microscopic method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 21/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50μm sections am made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.     

A simple method using alizarin red S for the detection of calcium in epoxy resin embedded tissue

This report presents a simple procedure for staining 1-2 microns epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue O solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralization. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated. Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.     

A Golgi-electron microscope method for insect nervous tissue.

Golgi's light microscope method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 2 1/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50 mum sections are made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.     

Hypophysis effect on the topographical distribution of toad oviduct mucoprotein components

In the present work the effects of the hypophysis hormones on oviduct mucoprotein components distribution patterns were studied. Remarkable changes after treating the toad with hypophysis injections were apparent. The distribution pattern for hexose, sialic acid, hexosamine and phosphate from 18 hours hypophysis treated toads were found to be identical with those obtained from preovulatory period animals. On the other hand, the levels for mucoprotein components from hypophysis treated animals were found to be approximately one-half or more higher than those obtained from postovulatory period toads. Otherwise, hypophysis treatment of the toads in preovulatory period had not effect on the levels and distribution patterns of mucoprotein components. These results suggest that hypophysis hormones are involved in the increase of the oviduct secretory activity.     

A Simple Method Using Alizarin Red S For the Detection of Calcium in Epoxy Resin Embedded Tissue

This report presents a simple procedure for staining 1-2 μm epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue 0 solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralition. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.     

Improved preservation of alkaline phosphatase in salivary glands of the cat

Synopsis The effects of tissue preparation on the localization of alkaline phosphatase were assessed at the light and electron microscopical levels. Glutaraldehyde-containing solutions were more inhibitory than formaldehyde alone but appeared to give betterin situ immobilization of the enzyme. The inhibitory effects of glutaraldehyde solutions were related especially to temperature and time and not necessarily to material absorbing at 235 nm. Distilled glutaraldehyde was the only form of glutaraldehyde to give consistently good results with least inhibition. Snap-freezing of pre-fixed tissue, after washing in a sucrose-containing solution, gave satisfactory results without undue ice-crystal formation. Immersion in dimethylsulphoxide before snap-freezing gave less good localization with greater diffusion of reaction product. Use of a Sorvall tissue-chopper on unfrozen tissue did not produce satisfactory sections. Free-floating sections of pre-fixed material appeared less inhibited than glass-mounted sections. The most satisfactory results were obtained after per-arterial perfusion with a 1% distilled glutaraldehyde-0.8% formaldehyde mixture, containing dextran, for up to 5 min followed by formaldehyde-dextran. The results were uniform; there was stronger staining of stromal surfaces of myoepithelial cells than previously, basal duct cells were also stained and occasional staining between acinar cells was now evident for the first time.