Luxol Fast Blue Combined With the Periodic Acid-Schiff Procedure for Cytological Staining of Kidney

Rat kidneys fixed in Regaud's fluid were stained by luxol fast blue (LFB), by the periodic acid-Schiff (PAS) method, and by LFB combined with PAS. When used separately the PAS stains the brush border, hyaline droplets and basement membranes reddish, the LFB stains the mitochondria, hyaline bodies and basement membranes greenish-blue. The combined LFB-PAS method stains the brush border reddish and the mitochondria dark blue, while the hyaline bodies and basement membranes are purplish colored. The LFB-PAS method provides color contrasts which show cytological features that are particularly significant in the kidney.     

Simplified Aldehyde-Fuchsin Staining of Neurosecretory Cells

Gomori's original aldehyde-fuchsin method has been modified by the combination of Halmi's counter stain with Gabe's preparation, consisting of basic fuchsin, 1 gm; boiling water, 200 ml; with HC1, 2 ml and paraldehyde, 2 ml added after cooling and filtering. The solution so made was allowed to ripen 3-4 days at room temperature, and the precipitate which formed was filtered off and dried at 55-60°C. The staining solution consisted of 0.5 gm of the dry precipitate dissolved in 100 ml of 70% alcohol. The staining follows original procedures except that it is very important to bring slides from water to 70% alcohol before placing them in the aldehyde-fuchsin solution and also to remove all excess staining solution by rinsing in 95% alcohol after staining. The staining solution is stable for at least 6 mo.     

Adaptation of the Millon, Sudan Black B, and Periodic Acid-Schiff Technics for Block Staining of Insect Tissues

Spermatophores and reproductive systems of the beetle, Lytta nuttalli Say, fixed in Bouin's aqueous picroformol or buffered 10% neutral formol were stained in toto by the Millon, Sudan black B and periodic acid-Schiff reactions as follows. Millon: after excess fixative is removed in 70% ethanol, specimens are brought to water, stained in Millon's reagent at 60 C for 1 hr, rinsed in 2% aqueous nitric acid at 40-50 C, dehydrated rapidly, cleared, embedded and sectioned as usual. Sudan black B: specimens are taken to absolute ethanol, stained in a saturated solution of Sudan black B in absolute ethanol at room temperature for 24-48 hr, rinsed and cleared in xylene, embedded and sectioned. PAS: specimens are brought to water, oxidized in 0.5 aqueous HIO₄ at 37 C for 30 min, washed in 2 changes of water, stained in Schiif reagent at room temperature for 1 hr, rinsed in 3 changes of 0.5% aqueous potassium metabisulfite, washed in running water for 10-15 min, dehydrated, cleared, embedded and sectioned. All 3 methods produced their characteristic staining in specimens up to 3 mm thick     

The Lipids Stained by the Periodic Acid-Schiff Technic

Unsaturated periodic acid-Schiff (PAS) stainable lipids of renal basement membranes are soluble in lipid solvents and do not add to the PAS staining in paraffin embedded sections. These lipids contribute to the staining of basement membranes in frozen sections. Pure sphingomyelin is stained by the PAS method if the oxidising solution is sufficiently acid and the time allowed for periodic oxidation is sufficient. This staining is considered to depend on splitting of the amide link between sphingosine and the fatty acid which leaves the 1-amino-2-hydroxyl grouping of sphingosine available for reacting with the periodic acid.     

The Lipids Stained by the Periodic Acid-Schiff Technic

Unsaturated periodic acid-Schiff (PAS) stainable lipids of renal basement membranes are soluble in lipid solvents and do not add to the PAS staining in paraffin embedded sections. These lipids contribute to the staining of basement membranes in frozen sections. Pure sphingomyelin is stained by the PAS method if the oxidising solution is sufficiently acid and the time allowed for periodic oxidation is sufficient. This staining is considered to depend on splitting of the amide link between sphingosine and the fatty acid which leaves the 1-amino-2-hydroxyl grouping of sphingosine available for reacting with the periodic acid.     

Application of the Periodic Acid-Schiff Procedure to Tissue Blocks

The periodic acid-Schiff procedure can be used for staining en bloc by incorporating the periodic acid with the fixing fluid. After simultaneous fixation and oxidation for 48 hr at room temperature and subsequent staining in Schiff reagent the tissues are dehydrated, embedded in paraffin and sectioned. Of two fixatives used, 95% alcohol proved superior to 10% formalin. Various concentrations of periodic acid (0.1-2.0%) yielded equally good results, thus the use of the lower concentrations is feasible and preferable. Fixation and oxidation simultaneously or separately yielded equally satisfactory results and in view of the time saved in the simultaneous method the authors recommend it. Using similar time of fixation and oxidation, satisfactory results were obtained with the intestine of rat after 3 hr of exposure to Schiff reagent. A longer period of exposure (up to 48 hr) was needed for comparable results with the kidney and liver.     

Application of the Periodic Acid-Schiff Technique to Whole Chick Embryos

The practicability of applying histochemical reactions to bulk staining has been explored by subjecting whole chick embryos at early stages to the periodic acid-Schiff (PAS) reaction. A comparison of the microscopic distribution of PAS positive substances revealed by this procedure with that obtained by the standard routine, i.e., staining of deparaffinized sections on slides, has shown similar localizations of PAS positive material and, in addition, finer morphological detail and more intensive reactions by staining the specimens in toto. The following method is recommended for chick embryos between stages 11-17 (Hamburger and Hamilton): Fixation in Gendre's fluid at 4°C; oxidation with alcoholic buffered periodic acid, 15 min; rinsing in distilled water, 10 min; Schift's reagent, 30 min; 3 sulfite rinses, 5 min each; running tap water, 10 min; dehydration, clearing and double-embedding in celloidin and paraffin.     

Staining of Pathogenic Fungi with Gomori's Aldehyde-Fuchsin in Both Old and New Sections

Gomori's stain for elastic tissue can be applied directly to paraffin sections as a routine method for fungus capsules and membranes. Either freshly prepared, or old sections previously stained with hematoxylin-eosin, can be used. After applying the aldehydefuchsin stain, nuclei may be stained (or restained) with hematoxylin, and a general background stain obtained with either eosin or light green.     

Differentiation of Mastocyte Granules by Tartrazine Counterstaining After the Periodic Acid-Schiff Procedure

When the periodic acid-Schiff stain is followed by a counterstain of 1% tartrazine O in 1% acetic acid, mastocyte granules turn to a brick-orange color. Pharyngeal and intestinal mucus remain unchanged and gastric mucus is only slightly affected. The best results were obtained after fixation in Cajal-De Castro fluid (95% ethanol, 50 ml; distilled water, 50 ml; chloral hydrate, 5-10 gm; and nitric acid, 5 ml).     

Demonstration of Alkaline Phosphatase and Periodic Acid-Schiff Positive Material in the Same Section

Alkaline phosphatase activity and periodic acid-Schiff (PAS) positive material were demonstrated in the same section by producing a gold-toned silver deposit at the sites of enzyme activity, followed by the PAS reaction. The glycerophosphate incubating mixture was modified by substituting AgNO₃ (final concentration about 0.17%) for the magnesium salt, and, after a slightly lengthened incubation, the silver was reduced by means of a fine grain photographic developer. Gold toning was followed by the regular PAS staining procedure. Gold deposits in the sites of enzyme activity were fully resistant to the action of periodic acid.     

Gross Demonstration of the Mammalian Atrioventricular Bundle by A Periodic Acid-Schiff Procedure

The following procedure stains the atrioventricular conduction system selectively. (1) Wash the fresh heart with physiological saline solution to free it of blood; (2) fix it in 10% formalin containing 0.5% HIO₄ for 1 hr; (3) wash in 3 changes of distilled water for 20 min; (4) keep in 80% alcohol for 12 hr to 2 wk; (5) wash with distilled water; (6) treat with a dilute Schiff's reagent containing 0.1 gm of basic fuchsin per 100 ml for 0.5-2 min; (7) rinse in three changes of 2% Na₂SO₃ in 0.2 N HCI for 3-5 min; (8) wash and examine in 80% alcohol; store in 80% alcohol.     

A Technic for Staining Mouse Pituitary

Many technics for histological staining of the pituitary gland have been devised. Since most of the methods described have been for animals other than the mouse, and since the mouse hypophysis, for some unknown reason, is difficult to stain by the accepted technics, the present writer has worked out a modification of Mallory's triple staining method.     

A Rhodocyan Technic for Staining the Anterior Pituitary

A reproducible, one-step, differential staining technic which uses routine formalin-fixed tissue and gives brilliantly contrasting results is produced by incubating sections for 1 hr in a 60° C oven in the following dye mixture: 1% eosin B (CI#771), 8 ml; 1% anilin blue (CI#707), 2 ml; and buffer solution (0.1M citric acid, 1.1 ml; 0.2M Na₂HPO₄, 0.9 ml; distilled water, 28.0 ml) at pH 4.5. No differentiation is necessary. The method can be modified for duodenal enterochromaffin cells and alpha cells of pancreatic islets by adjusting the buffer to pH 3.6 and staining for only 3 min at 60° C.     

Combined Fixing, Staining and Mounting Media

A number of non-volatile, water-soluble substances can be added to the usual aceto-cannine fixing fluids. These inert substances do not alter the fixation image and serve as mounting media when the volatile ingredients of the mixture evaporate. Formulae are given for solutions containing dextrin, dextrose, gelatin, pectin, sorbitol, and sucrose. Gum arabic can be incorporated in a formic-acid-carmine fixative. The limiting factor in the use of such mounting media in fixing fluids is the osmotic value they give the solution; with certain precautions, however, they can be used in place of the usual aceto-carmine treatment. The indices of refraction of these media are not as high as those of the natural balsams and the fixation images which the mixtures produce have the characteristic limitations of those secured by the aceto-carmine technic. Some of the natural balsams (Canada balsam, sandarac and Venetion turpentine) can also be incorporated in fixing fluids. These fixatives are able to hold balsam and water in solution together, and, as the volatile components of the mixtures evaporate, the fixed specimens remain in permanent balsam mounts. The addition of carmine to these fluids enables us to fix, stain, dehydrate, clear and mount a specimen in a single operation. These fixatives preserve more details of chromosome structure than the aceto-carmiae fluids, but their use is more limited; and they can be substituted for the latter only with certain favorable material, e.g., pollen mother cells of Rhoeo and Tradescantia and salivary gland chromosomes of Cbirono-mus. Some of the newer synthetic resins can be substituted for the natural balsams. Formulae are given for fixatives which contain Venetian turpentine, sandarac, Canada balsam and two synthetic resins.     

Combined Stain for Fish Pituitary

A new combined stain is described for the study of cell types in the fish pituitary. Tissues are prepared by fixing in formol-sublimate and then embedded in win wax. Tissue is sectioned at 5 μm and then stained sequentially with performic acid-alcian blue, periodic acid-Schiff, orange G, and acid fuchsin As a result of this procedure acidophils stain as follows: lactotropes, red; corticotropes, light pink melanotropes, bright pink and somatotropes, orange. Cyanophils stain either magenta red (gonadotropes) or blue (thyrotropes). Neurosecretory material and the fibers of the pars nervosa which penetrate the pars intermedia stain light blue.     

A Method for Combined Gross Skeletal Staining and Feulgen Staining of Embryonic Chick Tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

A Combined Alkaline Phosphatase-Pas Staining Method

Gomori's calcium phosphate method for alkaline phosphatase has been combined with the periodic acid-Schiff reaction on the same section. Paraffin sections of both formalin and alcohol fixed tissues have been employed. The technic is recommended as a selective staining procedure rather than a specific histochemical method. The Gormori alkaline phosphatase-PAS method has been compared with the silver alkaline phosphatase-PAS method developed by Moffat.