Pe. Reid et al., MECHANISM OF CONNECTIVE-TISSUE TECHNIQUES .1. THE EFFECT OF DYE CONCENTRATION AND STAINING TIME ON ANIONIC DYE PROCEDURES, Histochemical Journal, 25(11), 1993, pp. 821-829
Anionic dye connective tissue procedures were performed by staining fo
r 5 min and 24 h with (a) 0.00018 M and 0.0018 M solutions of 28 dyes,
and 0.018 M solutions of 21 dyes in saturated picric acid (SPA), and
(b) 0.0018 M and 0.018 M solutions of 20 dyes in 1% (w/v) phosphomolyb
dic acid (PMA). The staining obtained with dyes in SPA was classified
as selective (no cytoplasmic staining), moderately selective (traces o
f cytoplasmic staining) and non-selective (all other staining patterns
). The staining of collagen and cytoplasm with dyes in PMA was separat
ely classified on a scale of 1-5 (1 = no staining, 5 = maximum stainin
g). The selectivity of the staining obtained with SPA with solutions o
f dyes at concentrations of 0.00018 M and 0.0018 M, and both staining
times, was correlated (p < 0.001) with an empirical sulphonic acid con
stant (SAC) defined as the (number of dye sulphonic acid groups/dye mo
lecular weight) x 10(3). Correlation with molecular weight was poor an
d was significant only when staining was performed with 0.00018 m dye
solutions for 24 h. The dyes were divisible into three groups: group 1
(selectivity independent, or almost independent of staining time), gr
oup 2 (selective to moderately selective when staining was performed f
or 5 min), and group 3 (non-selective). The SAC of the group 1 dyes di
ffered significantly from those of the group 2 and 3 dyes. Selectivity
was essentially lost at dye concentrations of 0.018 M. The staining w
ith acidic dyes (no amines or substituted amines) in PMA differed sign
ificantly (p < 0.001) from that obtained with amphoteric dyes (contain
ing basic substituents). In general, acidic dyes stained cytoplasm. Am
photeric dyes with the exception of indigocarmine stained collagen. Ho
wever, most of these dyes also stained cytoplasm. In contrast to the r
esults obtained with dyes in SPA, selectivity correlated strongly with
molecular weight and only poorly with the SAC. Staining time and dye
concentration affected selectivity only when the acidic dyes were used
for 5 min at concentrations of 0.0018 M and 0.018 M. The data obtaine
d do not permit a clear distinction between the rate control and chemi
cal affinity models for the mechanism of staining with anionic dyes. H
owever, it seems possible that different groups of dyes stain by diffe
rent mechanisms.