The Glyoxal BIS(2-Hydroxyanil) Method Modified for Localizing Insoluble Calcium Salts

TO enable staining of insoluble calcium salts with glyoxal bis(2-hydroxyanil) (GBHA), the original solution containing 2 ml of 0.4% GBHA in absolute ethanol, and 0.3 ml of aqueous 5% NaOH, and limited to staining only soluble calcium salts, was modified as follows: 1, 2 ml of 0.4% GBHA in absolute ethanol in 0.6 ml of 10% aqueous NaOH; 11, 0.1 gm GBHA in 2 ml of 3.4% NaOH in 75% ethanol. To prevent diffusion and loss of calcium, the tissues were processed by the freeze-substitution or freeze-dry method and sections stained without removing the paraffin. Modification I is effective only when 1 or 2 drops placed on the section are evaporated gradually to dryness, concentrating the GBHA and NaOH on the insoluble calcium salts. Modification II is effective when dried or poured on the the section and allowed to stain for 5 min. The stained slides are immersed for 15 min in 90% ethanol saturated with KCN and Na₂CO₃ for specificity to calcium; rinsed and counterstained in 95% ethanol containing 0.1% each of fast green FCF and methylene blue; rinsed and dehydrated in ethanol; deparaffinized and cleared in xylene; and mounted in neutral synthetic resin. Although the modified methods tested on models failed to stain reagent grade CaCO₃ and Ca₃(PO₄)₂ crystals completely, apatite in developing vertebrae and calcified plaques in soft tissues were stained intensely red. The distribution of gross deposits of insoluble calcium salt in tissue sections corresponded with that shown in adjacent sections by the alizarin red S, ferrocyanide, and von Kossa methods. The modified GBHA method revealed smaller quantities of insoluble as well as soluble calcium salts discretely within cells where the other methods failed; also, calcium in cytoplasm of hypertrophied cartilage cells of developing vertebrae, and in cytoplasm of renal tubular cells of magnesium-deficient rats, not described previously, was demonstrated.     

Calcium in Cells of Fresh Bone Stained with Glyoxal Bis(2-Hydroxyanil)

Fresh ileum of adult rats and vertebrae and calvariae of newborn rats were immersed in a staining solution containing 0.1 gm of glyoxal bis(2-hydroxyanil) (GBHA) per 2 ml of 3.4% NaOH in 75% ethanol, dehydrated in absolute ethanol, cleared in xylene, and embedded in paraffin. Paraffin sections of stained material, 7 μ thick, were affixed to albumenized slides, immersed in 90% ethanol saturated with Na₂CO₄ and KCN to ensure specificity for calcium, rinsed in 95% ethanol, counterstained in 50% ethanol containing 0.1% methylene blue, dehydrated in absolute ethanol, deparaffinized and cleared in xylene, and mounted in neutral synthetic resin. By this procedure, red Ca-GBHA granules were deposited in goblet and Paneth cells, and in the cytoplasm of osteoblasts, osteocytes, chondrocytes, and periosteal cells of developing bones. Calcium in apatite did not stain. In osseous tissues sectioned in a cryostat or processed by the freeze-dry or freeze-substitution method, epiphyseal chondrocyte calcium was removed, and apatite stained so intensely red that it obscured calcium in the bone cells. Failure of control osseous tissues to stain after immersion in a 1% solution of disodium salt of ethylenediaminetetraacetic acid or in the alcoholic, alkaline solvent of the GBHA solution, indicated that the red granules in the cells of developing bone were due to calcium present in the cells in vivo and not due to absorption of GBHA by tissue components other than calcium, or to diffusion of Ca⁺⁺ during staining. Calcium localized in the cytoplasm and processes of the osteogenic cells suggests the need to re-evaluate the role of osteoblasts as depositors of calcium during osteogenesis.     

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.     

A Durable Nissl Stain for Frozen and Paraffin Sections

Frozen sections, 25-50 /j. thick, of formalin-fixed nervous tissues are mounted following the Albrecht gelatin technic. Paraffin sections, 15 p., are deparaffinized and transferred to absolute ethanol. The slides are then coated with celloidin. Both frozen and paraffin sections subsequently follow the same steps: absolute ethanol-chloroform (equal parts) for at least 20 min, 95% ethanol, 70% ethanol (1-3 min), then rinsed in distilled water. Sections are stained in Cresylechtviolett (Chroma) 0.5% aqueous solution containing 4 drops of glacial acetic acid per 100 ml, rinsed in distilled water, agitated in 70% ethanol until excess stain leaves the slide, and rinsed in 95% ethanol. Sections are then dehydrated in absolute ethanol, followed by butanol, cleared in xylene, and enclosed in permount.     

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

Summary 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 g/cm². The method allows detection of amounts of ionized calcium as low as 0.15 g/cm² or 0.0015 pg/².For the measurement of calcium in pancreatic tissue of fed rats, the tissue was subjected to freeze-substitution at –80°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.     

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.     

Staining Sulfated Mucopolysaccharides in Sections by Means of Acriflavine

Acriflavine gave insoluble salts with sulfated esters. Frozen or paraffin sections (fixed in 10% formol or Carnoy's solution) were stained in M/20 acriflavine solution and excess dye was rinsed in 95% alcohol. Then nuclei were stained with Meyer's haemalum. Thereafter the sections were washed in water, dehydrated in alcohol, cleared in xylene and mounted in balsam. Sulfated esters in the tissue sections were colored yellow or orange-yellow, generally more densely in frozen than in paraffin sections.     

Sequential Use of Wright's and Ziehl-Neelsen's Stains for Demonstrating Phagocytosis of Bacterial Spores

Nongerminating spores, germinating spores, and vegetative cells of Clostridium botulinum type A were observed during phagocytosis in the peritoneal fluid of white mice. Since phagocytes are easily ruptured by heat, the method described avoids heating, as this has been employed in conventional spore staining methods. A thin smear of the fluid is air dried on the slide for 2 hr, and stained by Wright's method: stain, 2 min; dilution water, 2 min; and rinsed; then in 0.005% methylene blue for 30 sec, and rinsed. This is followed by Ziehl-Neelsen's stain for 3-4 min and destained with 1: acetone-95% ethanol for 10 sec. The slide is rinsed, and Wright's staining repeated: stain 1 min, dilution 2-3 min; and thereafter washed about 5 ml of Wright's buffer. Blotting and air drying completes the staining. Non-germinating spores stain light red with a red spore wall, germinating spores are deep red throughout, vegetative cells are blue, and leucocytes show a dark purple nucleus and light blue cytoplasm.     

Staining Plant Tissues with Chlorazol Black E and Pianese III-B

The staining schedule was developed for a study of the mycorrhizae of red pine, Pinus resinosa Ait. From 70% alcohol, sections are stained in a saturated solution of chlorazol black E in 70% alcohol, 10-30 min; free dye removed by washing in 95% alcohol; stained 18-24 hr in Pianese III-b; rinsed in 95% alcohol, acidified by the addition of 2 ml of saturated aqueous picric acid per 100 ml, 3-4 changes or until the last change is pale yellow or light green; and rinsing in 95% alcohol to remove the acid. If the acid fuchsin is too intense, a cautious differentiation with 95% alcohol containing 1-3% of a 0.1 N solution of NaOH is made. If too much chlorazol black is removed, the effect can be compensated by overstaining with this dye at the beginning of the process. Sections are dehydrated, cleared, and covered in the usual manner. This stain has applications to plant tissues generally, and is particularly effective for meristematic tissues. It shows details of cytoplasmic structures and gives sharp delineation of primary cell walls.     

Stains recently certified

For staining in toto, planarians are fixed in a mixture of 10 ml of commercial formalin, 45 ml of 95% ethanol and 2 ml of glacial acetic acid. After treatment with 70% ethanol 3-10 days, they are washed in distilled water and immersed in 10% CuSO₄. 5H₂O for 3 hr at 50° C, transferred without washing to 1% AgNO₃ for 1.0-1.5 hr at 50° C; and then developed in: 10 ml of 1% pyrogallol, 100 ml of 56% ethanol and 1 ml of 0.2% nitric acid. Gold toning, 5% Na₂S₂O₃ and dehydration follow as usual. For staining sections, material is fixed in the same fixative, embedded in paraffin and sectioned at 10 μ. After bringing sections to water, they are immersed in 20% CuSO₄. 5H₂O for 48 hr at 37° C; then rinsed briefly in distilled water and placed in 7% AgNO₃ for 24 hr at 37° C. They are washed briefly in distilled water and reduced in: hydroquincne, 1 gm; Na₂SO₃, 5 gm and distilled water 100 ml. Gold toning, followed by 5% Na₂S₂O₃ and dehydration completes the process. Any counterstaining may follow.     

Gallocyanin-Chrome Alum Counterstaining of Golgi-Kopsch Impregnations

A simple technique is described for counterstaining Golgi-Kopsch impregnations. The sections are first stabilized by the method of Geisert and Updyke and then stained in 0.15% gallocyanin and 5% chromium potassium sulfate for 45 minutes at 55-60 C. The sections are then rinsed, dehydrated to 70% ethanol, cleared in terpineol, mounted and coverslipped. This procedure results in a light to medium blue stain of those cells not impregnated by the silver chromate. The major advantages of this procedure over earlier methods are: (1) the counterstain does not fade and (2) since no differentiation is required, many sections may be stained simultaneously.     

Gallocyanin-chrome alum counterstaining of Golgi-Kopsch impregnations

A simple technique is described for counterstaining Golgi-Kopsch impregnations. The sections are first stabilized by the method of Geisert and Updyke and then stained in 0.15% gallocyanin and 5% chromium potassium sulfate for 45 minutes at 55-60 C. The sections are then rinsed, dehydrated to 70% ethanol, cleared in terpineol, mounted and coverslipped. This procedure results in a light to medium blue stain of those cells not impregnated by the silver chromate. The major advantages of this procedure over earlier methods are: (1) the counterstain does not fade and (2) since no differentiation is required, many sections may be stained simultaneously.     

Staining Raw and Cooked Apple Tissues for Study of Cell Wall Structure

Permanent preparations were made of paraffin sections from raw and cooked apple tissues stained with microchemical color reagents for pectins and pentosans. Sections stained with ruthenium red to show pectins were dehydrated and covered in balsam, and sections stained with diphenylene diamine acetate (DDA) to show pentosans were washed with water and covered in Clearcol.

Cooking was accomplished by steaming cubed histological samples. Both raw and steamed specimens were fixed in FAA in a vacuum chamber, dehydrated and cleared in tertiary butyl alcohol, and embedded in paraffin. Paraffin sections first fixed to slides with Haupt's adhesive were further stabilized by immersing in a 1% celloidin solution after dissolving the paraffin.

Ruthenium oxychloride flakes were dissolved in a Coplin jar of water containing 2 drops of ammonium hydroxide. Rehydrated sections were stained in ruthenium red 30 minutes and rinsed in water. Three methods of further preparation follow: (1) Flood sections with 10% gum arabic; drain and air-dry thoroughly; immerse in xylene 5 minutes; cover in balsam. (2) Drain and air-dry sections; if desired, counterstain dry sections with Johansen's fast green solution; immerse in xylene; cover in balsam. (3) Dehydrate by dipping in 70%, 95%, and absolute ethyl alcohol; immerse in xylene; cover in balsam.

DDA was made by heating 15 g. of benzidine in 150 ml. of glacial acetic acid and 450 ml. of water until dissolved, then adding water to make 750 ml. of solution. Rehydrated sections were stained 4 hours in DDA, washed, stained 5 minutes in Congo red (Congo red, 5 g.; NaOH, 5 g.; water, 100 ml.), washed, and covered in Clearcol.

An Autotechnicon was used for dehydration, clearing, infiltration, deparaffinizing sections, and staining. Procedures that necessarily remained manual were fixation in a vacuum chamber, and all operations that followed staining.

Ruthenium red, though the best available indicator for pectins, may not be specific for these substances. DDA and ruthenium red stained identical structures in hypodermis and cortex. DDA also stained cuticle, hence was more useful than ruthenium red for delineating that portion. DDA sections were better for photomicrography, and for measuring thickness of cell walls. Neither stain prevented the study of cell walls in polarized light.     

A Phloxine-Azure-Hematoxylin Sequence for Differential Staining of Cells in Pancreatic Islets

Sections of 6 μ from tissues fixed in Susa or in Bouin's fluid (without acetic acid) and embedded in paraffin were attached to slides with Mayer's albumen, dried at 37 C for 12 hr, deparaffinized and hydrated. The sections fixed in Susa were transferred to a I₂-K₁ solution (1:2:300 ml of water); rinsed in water, decolorized in 5% Na₂S₂O₃; washed in running water, and rinsed in distilled water. Those fixed in Bouin's were transferred to 80% alcohol until decolorized, then rinsed in distilled water. All sections were stained in 1% aqueous phloxine, 10 min; rinsed in distilled water and transferred to 3% aqueous phosphotungstic acid, 1 min; rinsed in distilled water; stained 0.5 min in 0.05 azure II (Merck), washed in water; and finally, nuclear staining in Weigert's hematoxylin for 1 min was followed by a rinse in distilled water, rapid dehydration through alcohols, clearing in xylene and covering in balsam or a synthetic resin. In the completed stain, islet cells appear as follows: A cells, purple; B cells, weakly violet-blue; D cells, light blue with evident granules; exocrine cells, grayish blue with red granules.     

Modification of the enamel maturation pattern by vinblastine as revealed by glyoxal bis(2-hydroxyanil) staining and 45calcium radioautography

Summary Patterns characteristic of enamel maturation can be visualized at the surface of the rat incisor by staining with glyoxal bis(2-hydroxyanil) (GBHA) and radioautography following ⁴⁵calcium injection. In this study, the effects of vinblastine on enamel maturation were monitored by these two methods. At 4 h after injection of vinblastine, the darkly-stained GBHA bands had widened incisally into the interband regions when compared to normal, control teeth. Radioautography at 5 min after calcium injection in vinblastine-treated animals (4 h) showed a modified maturation pattern of weaker labeling and less distinct banding. At 8 h after vinblastine injection, most of the enamel stained uniformly with GBHA, and bands and interband regions could not be resolved. Radioautography at 5 min after calcium injection showed that the 8 h vinblastine treatment removed the banding pattern, leaving only a weakly-labeled area. Vinblastine is known to destroy and prevent the formation and turnover of microtubules, and hence the formation of ruffled borders of ruffle-ended ameloblasts (Akita et al. 1983). The concomitant decrease in calcium incorporation implies that events taking place in relation to the ruffled border may affect calcium exchange or accretion within the enamel.     

Luxol Fast Blue Mbs: A Stain for Phase Contrast Microscopy

A staining method to increase the contrast of sectioned material for phase contrast microscopy is described. Two stock solutions of the stain are required. The first is made by dissolving 2 gm of luxol fast blue MBS in 100 ml of 95% ethanol. The second solution is made up of 4 ml of a 29% aqueous solution of FeCl₃, 95 ml of 95% ethanol, and 1 ml of concentrated HCl. The staining solution is made by mixing equal parts of the two solutions. Sections are deparaffinized and taken to 70% alcohol, stained for 1.5 hr, dehydrated, cleared and covered as usual.     

Histochemical,ultrastructural and X-ray microprobe analytical studies of localization of calcium in the mucous lining of the rat duodenum

Summary Localization of calcium in a rapid frozen and freeze substituted duodenum of normal, starved or calciumrepleted rat was examined using either of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, a sensitive histochemical calcium stain or electron microscopy. In normallyfed rats, a majority of absorptive cells of the duodenum showed numerous discrete red granular GBHA reactions, approximately 1 m or less in diameter, located primarily along their lateral plasma membranes and within intercellular spaces. Electron microscopy also revealed electron-dense granules, 30–100 nm in diameter, showing a similar distribution as the GBHA granules in the respective absorptive cells, and confirmed their absence in mitochondria and other intracellular compartments. Some of the absorptive cells located exclusively at the tip of each villus contained highly GBHA-reactive tubulo-vesicular structures extending throughout the cytoplasm. However, they displayed virtually no granular GBHA reaction. In these cells, electron microscopy revealed numerous electron-dense granules in the nucleus, mitochondria and in other unidentified organelles. X-ray microprobe analyses of ultrathin sections confirmed the presence of calcium within electron-dense granules associated with both types of absorptive cells. The number and intensity of all GBHA reactions fluctuated according to luminal calcium concentration. In calcium-repleted rats, strong GBHA reactions appeared in a narrow zone of lamina propria at the tip of the villus, overlaid, predominantly, with absorptive cells showing tubulo-vesicular GBHA reactions. these results suggest the existence of distinct types of absorptive epithelial cells in the rat duodenum, with respect to patterns of calcium localization which they display.     

Mercuric Bromphenol Blue Staining of Precipitin Lines in Agar

Lines formed by antibody-organ antigen reactions are stained particularly well by a modification utilizing the mercuric bromphenol blue (MBB) mixture of Mazia et al. (Biol. Bull., 104: 57-67, 1953). The agar covered slides are placed overnight in 0.85% NaCI at 4 C, followed by washing for 2 hr in 0.85% NaCI at 25 C. They are then rinsed for 10 min in distilled water, and dried overnight at 37 C. The precipitin lines are fixed by immersing the slides for 25 min in 95% alcohol, followed by 5 min hydration in distilled water. They are stained for 25 min in MBB mixture (HgCI₂, 10 gm; bromphenol blue, 0.1 gm; 95% ethanol, 100 ml). Excess stain is removed by immersing in acidified alcohol (95% ethanol, 98 ml; glacial acetic acid, 2 ml). Finally, the slides are passed through alcohol and xylene, and resin-mounted under coverslips.     

A staining method of epoxy-embedded lymphatic and hemopoietic tissues in autoradiograms

Summary Autoradiograms of sections 0.8–1.0 thick of epoxy-embedded, glutaraldehyde and OsO₄ fixed lymphatic and hemopoietic tissues were stained with 4% aqueous basic fuchsin at 70°C for 3 min, rinsed well and destained twice at 70°C for 1 min. A 1% methylene blue solution dissolved in 1% borax aqueous solution mixed with 1% aqueous azur II solution in the proportion of 1:1 was then allowed to act on the slide at 70°C for 1 min. The stain was rinsed well off slide and destained also twice at 70°C for 1 min.The preparation was dried before applying a mounting medium (Eukitt) and cover glass. An Eukitt mounting prevented the stain from fading for at least 1 year. The method is simple, rapid and provides sharp contrast in autoradiography with well destained nucleal emulsion.This study was supported by Alexander von Humboldt-Foundation.     

Histochemical, ultrastructural and X-ray microprobe analytical studies of localization of calcium in the mucous lining of the rat duodenum

Localization of calcium in a rapid frozen and freeze substituted duodenum of normal, starved or calcium-repleted rat was examined using either of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, a sensitive histochemical calcium stain or electron microscopy. In normally-fed rats, a majority of absorptive cells of the duodenum showed numerous discrete red granular GBHA reactions, approximately 1 micron or less in diameter, located primarily along their lateral plasma membranes and within intercellular spaces. Electron microscopy also revealed electron-dense granules, 30-100 nm in diameter, showing a similar distribution as the GBHA granules in the respective absorptive cells, and confirmed their absence in mitochondria and other intracellular compartments. Some of the absorptive cells located exclusively at the tip of each villus contained highly GBHA-reactive tubulo-vesicular structures extending throughout the cytoplasm. However, they displayed virtually no granular GBHA reaction. In these cells, electron microscopy revealed numerous electron-dense granules in the nucleus, mitochondria and in other unidentified organelles. X-ray microprobe analyses of ultrathin sections confirmed the presence of calcium within electron-dense granules associated with both types of absorptive cells. The number and intensity of all GBHA reactions fluctuated according to luminal calcium concentration. In calcium-repleted rats, strong GBHA reactions appeared in a narrow zone of lamina propria at the tip of the villus, overlaid, predominantly, with absorptive cells showing tubulo-vesicular GBHA reactions. These results suggest the existence of distinct types of absorptive epithelial cells in the rat duodenum, with respect to patterns of calcium localization which they display.