Problems with hematoxylin staining:
(1) variation in the stock chemical (source, lot to lot, etc)
(2) oxidation of the stain during storage (prolonged exposure to sunlight, air, etc)
(3) incorrect concentration of the staining solution
(4) failure to filter the stain prior to use
(5) incorrect pH of the staining solution
(6) prolonged use of the stainng solution
(7) problems in tissue fixation or processing
(8) tissue overdecalcification
(9) incomplete deparaffinization of the section
(10) problems with the glass slides, including use of adhesives
(11) variation in section thickness
(12) temperature extreme during staining
(13) variation in timing of the staining steps
(14) failure to wash adequately prior to the stainng step
(15) problems in washing after the staining step (incomplete, chlorine in water, contaminants, etc)
(16) problems with decolorization if an overstaining (regressive) method is used
(17) problems with the alkaline bluing reagent (not used or wrong pH)
Once possible explanations are identified then the staining is repeated after making corrections. The staining quality should be good once all of the problems have been eliminated.
