New Uses for Calcium Chloride Solution as a Mounting Medium

Fresh cross sections of stems [Psilotum nudum, Coleus blumei, and Pelargonium peltatum] and roots (Setcreasea purpurea) 120 μm thick were fixed in FPA₅₀ (formalin: propionic acid: 50% ethanol, 5:5:90, v/v) for 24 hr and stored in 70% ethanol. The sections were transferred to water and then to 1% phloroglucin in 20% calcium chloride solution plus either hydrochloric, nitric, or lactic acid in the following ratios of phloroglucin-CaCl₂ solution:acid: 25:4, 20:2, or 15:5. The sections were mounted on slides either in one of the three mixtures or in fresh 20% calcium chloride solution. A rapid reaction of the acid-phloroglucin with lignin produced a deep red color in tracheary elements and an orange-red color in sclerenchyma. Fixed and stored leaf pieces from Nymphaea odorata were autoclaved in lactic acid, washed in two changes of 95% ethanol, transferred to water, and treated with the three acid-phloroglucin-calcium chloride mixtures. The abundant astrosclereids stained an orange-red color similar to that of sclerenchyma in the sections. In addition, a new method is reported for specifically staining lignified tissues. When sections or leaf pieces are stained in aqueous 0.05% toluidine blue O, then placed in 20% calcium chloride solution, all tissues destain except those with lignified or partially lignified cell walls. Thus, toluidine blue O applied as described becomes a reliable specific test for lignin comparable to the acid-phloroglucin test.     

Staining of Acid Mucopolysaccharides after Chromatography on Filter Paper

Acid mucopolysaccharides obtained both from commercial sources and by isolation from human urine have been chromatographed on Whatman No. 1 filter paper, using propanol or ethanol in pH 6.5 M/15 phosphate buffer as solvent systems. The chromatograms are then fixed by immersion in 95% alcohol and in diethyl ether. After drying, they are stained in 0.06% toluidine blue O in 0.5% aqueous acetic acid. A final rinsing in 2% aqueous acetic acid removes the excess dye from the paper and exposes the stained mucopolysaccharide to a pH favoring orthochromasia.     

Staining of Acid Mucopolysaccharides after Chromatography on Filter Paper

Acid mucopolysaccharides obtained both from commercial sources and by isolation from human urine have been chromatographed on Whatman No. 1 filter paper, using propanol or ethanol in pH 6.5 M/15 phosphate buffer as solvent systems. The chromatograms are then fixed by immersion in 95% alcohol and in diethyl ether. After drying, they are stained in 0.06% toluidine blue O in 0.5% aqueous acetic acid. A final rinsing in 2% aqueous acetic acid removes the excess dye from the paper and exposes the stained mucopolysaccharide to a pH favoring orthochromasia.     

Staining Mitochondria in Saccharomyces cerevisiae

After testing various procedures (amidoblack 10B, acid fuchsin-methyl blue, Luxol fast blue MBS-phloxine, toluidine blue O, Jams green B and pinacyanol), three stains can be recommended for staining both types of mitochondria (globose and threadlike) in the cells of Saccharomyces cerevisiae: (1) 0.1% solution of amidoblack 10B in citrate buffer (pH 3.0) for 10 min; (2) 0.01% solution of toluidine blue O in phosphate buffer (pH 6.0) for 30 min; (3) 0.01% solution of Janus green B in distilled water (pH 5.6) for 30 min. The latter stain is most specific because its staining reaction depends upon the action of the mitochondrial enzyme cytochrome c oxidase. Yet, low concentrations and short incubation periods must be applied to avoid poisoning of the cell metabolism.     

A silver-impregnation-toluidine blue technique for demonstration of embryonic skeletal structures in paraffin sections

Summary Dewaxed and hydrated sections are treated with 1% aq. silver nitrate in bright light for 60 min and then thoroughly washed with distilled water followed by a rinse in Kolthoff's buffer (pH 3.3). The sections are next treated with 0.25% toluidine blue dissolved in buffer for 3 min, rinsed in buffer, allowed to dry and then cleared and mounted. Foci of calcification appear black and cartilage-matrix lilac to deep mauve. Nuclei appear deep blue in contrast to the pale blue of the cytoplasm.     

Staining Tissue of the Central Nervous System with Luxol Fast Blue and Neutral Red

The tissue is fixed in 10% neutral saline formalin for 1 day to 3 wk depending on the size of the block, dehydrated and embedded in paraffin. The sections are stained at 57° C for 2 hr, then at 22° C for 30 min, in a 0.0125% solution of Luxol fast blue in 95% alcohol acidified by 0.1% acetic acid. They are differentiated in a solution consisting of: Li₂CO₃, 5.0 gm; LiOH-H₂O, 0.01 gm; and distilled water, 1 liter at 0-1° C, followed by 70% alcohol, and then treated with 0.2% NaHSO₃. They are soaked 1 min in an acetic acid-sodium acetate buffer 0.1 N, pH 5.6, then stained with 0.03% buffered aqueous neutral red. Sections are washed in distilled water, 1 sec, then treated with the following solution: CuSO₄·5H₂O, 0.5 gm; CrK(SO₄)₂·12H₂O, 0.5 gm; 10% acetic acid, 3 ml; and distilled water, 250 ml. Dehydration, clearing and covering complete the process. Myelin sheaths are stained bright blue; meninges and the adventitia of blood vessels are blue; red blood cells are green. Nissl material is stained brilliant red; axon hillocks, axis cylinders, ependyma, nuclei and some cytoplasm of neuroglia, media and endothelium of blood vessels are pink.     

A Rapid HCl/Toluidine Blue Squash Technic for Plant Chromosomes

Fresh or pretreated root tips are simultaneously fixed and hydrolysed in 5 N HC1 for 15 min at room temperature. After washing they are macerated on a slide in a drop of 0.05% toluidine blue made up in McIlvaine citric acid-Na₂HPO, buffer at pH 4.0. Pressure on the cover slip completes the squash preparation. It is made permanent by removing the cover slip on dry ice, air drying and mounting in Euparal.     

Staining of different endocrine cells with hydrochloric acid-toluidine blue in Epon embedded rat tissue

The usual HCl-toluidine blue staining of different endocrine cells is applicable to paraffin embedded material. A modification for Epon embedded tissue suitable for consecutive light and electron microscopic studies is described which makes it possible to find the same stained cell, both in a semithin section and in subsequent ultrathin sections. This method facilitates the search for scattered specific endocrine cells. Without removing the resin, sections of Epon embedded tissues were hydrolyzed for 17 hr in 1% HCl at 65 C and stained for 2 hr in 0.1% toluidine blue in McIlvaine buffer, pH 5.8. The following cells were stained: C cells in thyroid glands; A and D cells in pancreatic islets; B cells in anterior pituitary; G, D and Ec cells in the gastrointestinal tract; Ad cells of the adrenal medulla.     

Staining of Different Endocrine Cells with Hydrochloric Acid-Toluidine Blue in Epon Embedded Rat Tissue

The usual HCl-toluidine blue staining of different endocrine cells is applicable to paraffin embedded material. A modification for Epon embedded tissue suitable for consecutive light and electron microscopic studies is described which makes it possible to find the same stained cell, both in a semithin section and in subsequent ultrathin sections. This method facilitates the search for scattered specific endocrine cells. Without removing the resin, sections of Epon embedded tissues were hydrolyzed for 17 hr in 1% HCl at 65 C and stained for 2 turn 0.1% toluidine blue in McIlvaine buffer, pH 5.8. The following cells were stained: C cells in thyroid glands; A and D cells in pancreatic islets; B cells in anterior pituitary; G, D and Ec cells in the gastrointestinal tract; Ad cells of the adrenal medulla.     

Staining of Mucus with Buffered Solutions of Toluidine Blue O, Thionin and New Methylene Blue N

The addition of buffer mixtures to toluidine blue O, thionin and new methylene blue N improves their use in the staining of mucus after formaldehyde fixation. Overstaining is minimi The buffer mixtures used consisted of variable proportions of M/10 citric acid and M/5 anhydrous Na²HPO₄ in 25% methanol. Connective tissue mucus stained satisfactorily with these dyes at a buffer pH range of 3.4 and 3.95 and epithelial mucus at a range of 2.2 to 3.95. The corresponding ratios of M/10 citric acid: M/5 Na₂HPO₄ are 16:4 to 14:6 and 20 A to 14:6 respectively.     

The Peracetic-Orcein-Halmi Stain: A Stain for Connective Tissues

A selective stain useful for the study of connective tissues is described. The stain demonstrates elastic and oxytalan fibers as well as fibrils in mucous connective tissues previously undescribed. Reticular fibers are not stained. The stain may be used on sections that have been fresh frozen or fixed in formalin or ethanol. Sections are deparaffinized, washed in absolute ethanol, oxidized in peracetic acid 30 min, washed in running water, stained in Taenzer-Unna orcein 15 min, 37°C, differentiated in 70% ethanol, washed in running water, stained in Lillie-Mayer alum hematoxylin 4 min, blued in running water, and counterstained 20 sec in a modified Halmi mixture of 100 ml distilled water, 0.2 gm light green SF, 1.0 gm orange G, 0.5 gm phosphotungstic acid and 1.0 ml glacial acetic acid. Sections are rinsed briefly in 0.2% acetic acid in 95% ethanol, dehydrated and mounted.     

Ultrastructure of cardiac muscle of Trochosa terricola Thor., Pardosa amentata Clerck,P. pullata Clerck,and Pisaura mirabilis Clerck (Araneae: Lycosidae,Pisauridae)

Summary The ultrastructure of the cardiac muscle in some araneae has been investigated. The sarcolemma invaginates at the Z band and may extend into the cells through several myofibrils. Numerous T-tubules are given off both from sarcolemmal invaginations and also directly from the cell surface. In passing through the Z band, the luminal diameter of T-tubules greatly increases. Dyadic and a few triadic couplings are found mainly at the A-I level. Peripheral couplings were not seen.The ruthenium red solutions employed were prepared as described by Myklebust (1975), but the fixative contained 2% sucrose. The hearts were fixed for 3 1/2h in the ice cold solution, washed in buffer and post-fixed for 1 1/2h in a ruthenium red/OsO₄ solution. Dehydration and embedding was performed as described above. Sections were cut on a Reichert Om U2 microtome, stained with lead citrate (Reynolds, 1963) and examined in a Philips 300 electron microscope at 80 kV.Semithin sections for light microscopy were stained with toluidine blue (Mercer and Birbeck, 1972).I am much obliged to cand. real Erling Hauge for identifying the specimens used in the investigation proportion of relaxed sarcomeres. The fixed pieces were washed in a buffered sucrose solution and post-fixed in a 1% OsO₄ solution buffered with sodium cacodylate for 1 h. Following washes in a compatible buffer and in distilled water, the tissue was stained en bloc for l h in a 2% aqueous uranyl acetate solution. The tissue was dehydrated through cold acetone and embedded in Epon 812.     

Brazilin-Toluidine Blue O and Hematoxylin-Darrow Red Methods for Brain and Spinal Cord

Tissue fixed in 10% formalin, formalin-95% ethanol 1:s CaCO₂ or phosphate buffer neutralized formalin, or methanol-chloroform 2:1, was dehydrated and embedded in paraffin or double-embedded by infiltration in 1% celloidin followed by a chloroform-paraffin sequence. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 24-26 C. For either method, mordanting 30-60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. For brazilin-toluidne blue O, myelin was stained for 20-60 min, depending upon section thickness, in a self-differentiating solution consisting of: 0.15% Li₂CO₃ 75 ml; 6% brazilin in 95% ethanol, 25 ml; and NaIO₃ 75 mg. After a thorough washing, Nissl material was stained for 3-8 min in a solution consisting of: 0.1 M acetic acid, 90 ml; 0.1 M sodium acetate, 10 ml; and 1% toluidine blue 0, 2.5 ml. For hematoxylin-Darrow red, myelin was stained for 2-6 hr in a self-differentiating solution consisting of: 0.15% Li₂,CO₃ 95 ml; 10% hematoxylin in 95% ethanol, 5 ml; and NaIO₃ 25 mg. After a thorough washing, Nissl material was stained for 20 min or less in a solution consisting of: 0.1 M acetic acid, 90 ml; 0.1 M sodium acetate, 10 ml; Darrow red, 25 mg. This mixture was first boiled, cooled to room temperature and filtered. In both methods, washing, dehydration, clearing, and mounting completed the process. In the brazilin-toluidine blue technic, myelin sheaths were stained reddish purple; neuronal nuclei light blue with dark granules of chromatin; nucleoli dark blue; and cytoplasm blue with dark blue Nissl granules. In the hematoxylin-Darrow red procedure, myelin sheaths were blue-black; nuclei light red with dark granules of chromatin; nucleoli almost black; and cytoplasm red with bright red Nissl granules.     

Metachromatic Reaction of Pancreatic B Cells to Toluidine Blue; Influence of pH on Staining

The granules of islet B cells show an intense β metachromasia when paraffin sections of pancreas fixed in Bouin's fluid or formalin are dipped for 1 min in a 0.1% aqueous solution of toluidine blue O₂ buffered to pH 6.0 with acetate or phosphate. This reaction provides a quick method for surveying the condition of B cells in experimental work. A weak staining is observable at pH 4.5 and becomes distinct at pH 5.5-6.0. Oxidation of sections (0.25% KMnO₄ in 0.5% H₂SO₄, for 1 min, recommended) prior to staining intensifies the metachomatic reaction conspicuously. The metachromatic substance could not be demonstrated after fixation in either ethanol or acetone. It corresponds to the aldehyde fuchsin-positive and pseudoisocyanin-metachromatic substance in its occurrence and distribution in the B cells, as shown by different physiological states of various animals, including fasted and glucose-administered guinea pigs. It is thought to be topographically coincident but not necessarily identical to insulin.     

Enzyme Clearing of Alcian Blue Stained Whole Small Vertebrates for Demonstration of Cartilage

Preparation of small vertebrates cleared after alcian blue Staining of cartilage is facilitated by trypsin digestion. Specimens are fixed in formalin, washed, skinned, and eviscerated. After staining in a solution of alcian blue in acetic acid-alcohol for 24-48 hours, they are transferred to water through graded alcohols. Excess db blue is removed over a period of up to three weeks by changes every 2-3 days of 1% trypsin in approximately we-third-saturated sodium borate. Bony tissues may be stained after this in a solution of alixarin red S in 0.5% KOH. Specimens rue bleached if necessary and dehydrated through graded KOH-glycerine mixtures for storage in glycerine. Since alcohol treatment in addition to formalin fixation does not affect results with this method, it should be useful to researchers who want to study the cartilage or cartilaginous skeletons in museum specimens, which me routinely fixed in formalin and stored in alcohol.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.     

Selective Staining of Endocrine Cells by Basic Dyes After Acid Hydrolysis

Staining of tissue sections by basic dyes after immersion in hot hydrochloric acid (0.2 N for 3-10 hr at 60 C) provides a means for selective detection of many endocrine cells. The acid hydrolysis suppresses diffuse basophilia, mainly due to RNA, DNA and acid polysaccharides, and increases the basophilia of secretory granules in endocrine cells, due, at least in part, to the proteins they store. After such treatment, toluidine blue or azur A (0.01-0.005% in 0.02 M McIlvaine buffer, pH 5) or pseudoisocyanin (0.02% in distilled water) heavily stain A and D cells of pancreatic islets, enterochromaffin and nonenterochromaffin endocrine cells of the gastrointestinal mucosa, thyroid parafollicular or C cells, pituitary basophil cells and adrenalin-secreting cells of the adrenal medulla.     

Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage.

Preparation of small vertebrates cleared after alcian blue staining of cartilage is facilitated by trypsin digestion. Specimens are fixed in formation, washed, skinned, and eviscerated. After staining in a solution of alcian blue in acetic acid-alcohol for 24-48 hours, they are transferred to water through graded alcohols. Excess alcian blue is removed over a period of up to three weeks by changes every 2-3 days of 1% trypsin in approximately one-third-saturated sodium borate. Bony tissues may be stained after this in a solution of alizarin red S in 0.5% KOH. Specimens are bleached if necessary and dehydrated through graded KOH-glycerine mixtures for storage in glycerine. Since alcohol treatment in addition to formalin fixation does not affect results with this method, it should be useful to researchers who want to study the cartilage or cartilaginous skeletons in museum specimens, which are routinely fixed in formalin and stored in alcohol.     

Fixation and Staining of Planaria for Histological Study

Fixation and staining of planaria can affect the interpretation of histopathological changes following their exposure to various agents. We assessed several fixation protocols with various stains in planaria to determine an optimal combination. Planaria were fixed in each of the following: 10% neutral buffered formalin, 2.5%, glutaraldehyde, Bouin's, Zenker's, 70% ethanol, and relaxant. In addition, planaria were fixed in relaxant and postfixed in each of the fixatives above. Paraffin embedded sections from each fixation protocol were stained with hematoxylin and eosin (H & E), toluidine blue, periodic acid-Schiff (PAS), or phosphotungstic acld-hematoxylin (PTAH). Relaxant fixed planaria were also stained with Steiner's, Holmes, trichrome, Giemsa, Grocott's methenamine silver (GMS) and antibodies for intermediate filaments (cytokeratin, vimentin and desmin). Relaxant and Zenker's gave the best fixation with minimal artifacts. Formalin, glutaraldehyde, and ethanol were unacceptable because they caused contortions of the body, crenation, and a darkly pigmented epidermis. Gastroderm could be differentiated from stroma best when stained with H & E, toluidine blue and PTAH. Other organ systems differentially stained included the epidermis, marginal adhesion gland, nervous tissue, and muscle. PAS, Steiner's, Holmes, trichrome and the intermediate filament stains were not useful for planaria staining. The most morphological information was obtained with relaxant fixative and a combination of sections stained with H & E and PTAH.