A Six-Dye Staining Schedule for Sections of Mesquite and Other Desert Plants

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Abstract
Materials are fixed in FPA (formalin, 2; propionic acid, 1; 70% ethanol, 17). Paraffin sections on slides are brought to 50% ethanol and stained as follows: (1) in Bismarck brown Y, a 0.02% solution in 0.1% aqueous phenol, 10-30 min.; wash 30 sec. in 0.7% acetic acid, and wash in distilled water 20-30 sec; (2) in crystal violet, 1% in 70%, ethanol alkalinized with 1 drop of 1 N NaOH per 100 ml, 12-35 min.; wash 30-60 sec. in tap water to remove excess stain, and rinse 0.5 sec. in 70%, ethanol; then mordant in I2-KI, 1%- each in 70% ethanol, 40 sec., and rinse in 70% ethanol 2-5 sec.; (3) in a mixture containing 0.4% acid fuchsin and 0.6% erythrosin B in 70% ethanol about 0.5 sec.; rinse in 70% ethanol 5-15 sec. to remove excess red; dehydrate in 70%, 95%, and absolute ethanol, 2-3 sec. each; (4) in fast green FCG, 0.5% in a mixture of equal parts of methyl cellosolve, absolute ethanol, and clove oil, 5-15 sec; rinse in a mixture of clove oil, 10 ml; absolute ethanol, 100 ml; and methyl cellosolve, 10 ml, 5-7 sec.; (5) in orange G, 0.75 gm in a mixture of clove oil, 40 ml; absolute ethanol, 40 ml; and methyl cellosolve, 60 ml, b-30 sec.; rinse clean in a 1:1 mixture of xylene and absolute ethanol, 5-20 sec. Complete the clearing in pure xylene, 3 changes, 1.5 min. in each, and apply a cover glass with synthetic resin. Slides are agitated in all steps except Bismark brown Y, crystal violet, and the xylenes. Contrast and staining intensity are adjusted by varying staining times in the dye solutions.