R. D. Lillie

Histochemical Use of Borohydrides as Aldehyde Blocking Reagents

Complete blocking of the Schiff reaction applied after HIO4 oxidation is attained by 1%, 0.5% or 0.2% NaBH4 in 1% Na2HPO4 with a 1 min exposure, 0.1% NaBH4 requires 2 min. KBH4 was also completely effective in the same solvent at 1, 0.5 and 0.25% in 1 min. The solutions deteriorate on standing, so that 0.5% NaBH4 is effective in 1 min at 7 hr but 5 min is needed at 24 hr and is ineffective at 36 hr; 1% KBH4 requires 4 min at 24 hr when fresh, and showed deterioration in 2–4 days. Saturated (0.4%) isopropanol and 1% pyridine solutions required 10–15 min when fresh and showed deterioration in 2–4 days. No satisfactory results were obtained with 80% or absolute dioxan or with methyl cellosolve in times of an hour or less; even a 24 hr exposure was ineffective with 0.2% in 80% dioxan.

Histochemical Identification of Basic Proteins with Biebrich Scarlet at Alkaline pH

Staining at graded alkaline pH levels with the sulfonated dye, Biebrich scarlet, shows basic proteins in various histologic sites, and differentiates the sites according to their relative basicity. Certain structures stain at pH 6.0 but not at 8.0 or above. Others stain maximally up to pH 93 and a few stain strongly as high as pH 103. The most strongly basic sites resist more than the others the destruction of acidophilia by nitrosation, acetylation or exposure to formaldehyde.

Black periodic and black Bauer methods for tissue polysaccharides.

m-Aminophenol gives a more rapid and more complete blockage of the Schiff reaction of aldehydes produced in tissue by oxidation with periodic or chromic acid than does aniline. Moreover, the phenol thus attached to aldehyde sites in tissue can then be azo coupled with alkaline solutions of fresh or stabilized diazonium salts. When so used at pH 8, fast black K, C.I. 37190, shows glycogen, mucins, basement membranes, muscle stroma, fungus cell walls, etc. stained black, on a somewhat grayish pink to red background.

The Diazosafranin Method; Control of Nitrite Concentration and Refinements in Specificity

Safranin is diazotized by using the customary molar ratio—dye, 1:HCl, 3:NaNO2l. Partly oxidized NaNO2 can be used, if necessary, by increasing the concentration of its solution enough to cause the normal color change from red to deep blue to occur within 2 min after adding the NaNO2. To avoid carrying over excess HNO2 into the alkaline coupling solution, 1 ml of 3% alcoholic urea solution (30 mg) is added for each milliliter excess of 1 N NaNO2 used. Any free HNO2 remaining at the end of the diazotization period produces a deep blue violet on starch-KI paper. Prolonged acid washing may be applied after coupling to decolorize cationic dye staining or triazenes. Na2S2O4, TiCl3 or SnCl2 may be used to bleach true azo colorations. This decolorization is not limited to newly formed azo compounds with tissue.

Acid Orcein-Iron and Acid Orcein-Copper Stains for Elastin

Paraffin sections of formol-fixed tissues stained 4–18 hr in 70% alcohol containing 1% orcein and 1% of concentrated (12 N) HCl by volume yield the familiar purple brown elastin and red nuclei on a pink background. When sections so stained are transferred directly from the stain to 70% alcohol containing 0.02% ferric chloride (FeCl3·6 H2O) or 0.02% copper sulfate (CuSO4·5 H2O) for a 15 sec to 3 min period, elastin coloration is changed to black or reddish black and chromatin staining to reddish black. The procedure can be counterstained with picro-methyl blue to yield blue collagen and reticulum or with our flavianic acid, ferric chloride, acid fuchsin mixture to give deep yellow background and deep red collagen.

Difficulties in ferrous iron mordant hematoxylin staining and some hematoxylin substitutes.

A gradual deterioration of intensity of sequence ferrous sulfate hematoxylin staining was traced, after elimination of hematoxylin quality as a cause, to a deterioration of the metal salt, associated with caking of the crystals. Fresh samples were also partly caked and ineffective. Ferrous ammonium sulfate was found also subject to the same deterioration. Ferrous chloride freshly prepared as a 1 M solution from iron wire under anaerobic conditions at biweekly intervals proved to be satisfactory as a mordant source. Of several other mordant dyes tested: gallein, brazilin and chromoxane pure blue B were the best, but none was equal to good hematoxylin.

Safranin O: Lot Variation in Performance as a Diazonium Base

The performance of safranin O (C.I. 50240) as a diazonium base was tested on 24 lots with Biological Stain Commission certification dates ranging from 1931 to 1972. In general, recently certified lots performed better than the oldest lots, but good performance was obtained with lots certified in 1951-52. Dye content specified for certification tests did not correlate with performance as a diazo. Differences in average performance of samples from the 4 suppliers showed less variation than the variation shown by individual lots from some of the suppliers; moreover, these variations did not show a close correlation attributable to the age of the sample. Spectroscopy revealed a smaller group with λ = 530-532 nm and a larger one at 534, but there was no consistent variation in average performance as a diazo reagent attributable to the absorption maxima.