A rapid safranin-metal phthalocyanine double staining technique for plants

Pure metal 4,4',4'',4'''-tetra-substituted, sulfo-, carboxy- and nitrophthalocyanines were synthesized. Mounted, deparaffinized and partially dehydrated sections of plant tissues were stained with 0.5% safranin in 50% alcohol for 5-10 min. Excess safranin was removed with a series of 70%, 95% and absolute alcohol washes. The sections were then stained for 2-3 min using metal 4,4',4'',4'''-phthalocyanine tetracarboxylic acid (MPTC, 0.5% (V/V) containing a few drops of dilute sodium hydroxide), metal 4,4',4'',4'''-tetrasulfophthalocyanine (MPTS, 0.5% (V/V)) or metal tetranitrophthalocyanine (MPTN, 0.5% (V/V) in dimethyl sulfoxide). The sections were washed with 95%, then absolute alcohol; however, the metal tetranitrophthalocyanine section was washed only with absolute alcohol. Stained sections were treated briefly with xylene, then mounted on a coverslip. Bright peacock blue (MPTC and MPTS using Cu, Co or Ni), turquoise blue (MPTN using Cu or Ni) or parrot green (zinc phthalocyanine tetracarboxylic acid-ZnPTC, zinc phthalocyanine tetranitro derivative-ZnPTN) colors were obtained. Lignin-containing cells were stained red by safranin and the remaining cell structures were stained by the metal phthalocyanine complex with color brightness superior to that of fast green. Uniform staining, no color fading after a year, reliability, brief staining times, high color contrast (log epsilon = 4.0-4.9) and ease of use make this double staining combination ideal for routine use and photomicrography.