Routine (H&E) and Special Staining
H&E chemistry
Hematoxylin is a basic dye that binds acidic nucleic acids, staining nuclei blue-purple; eosin is an acidic dye that binds basic proteins, staining cytoplasm, collagen, and red cells pink. Progressive hematoxylin (Gill, Mayer) needs no differentiation; regressive hematoxylin (Harris) requires acid-alcohol differentiation followed by bluing in ammonia water, lithium carbonate, or Scott's tap water. Over-differentiation leaves pale nuclei; under-differentiation leaves cloudy blue cytoplasm. Eosin overstaining obscures nuclear detail.
Special stains
Special stains depend on specific chemistry and require controls. PAS stains glycogen, mucin, and basement membranes magenta; diastase digests glycogen and serves as the control. Masson trichrome stains collagen blue or green and muscle red, but variants differ. Reticulin is a silver stain (black fibers), Prussian blue detects iron, Congo red shows amyloid with apple-green birefringence, GMS stains fungi black, acid-fast stains are red, and Alcian blue highlights acid mucins. Always know the expected control result and the counterstain.
Traps and a worked example
Distractors include confusing trichrome color schemes, forgetting diastase, incomplete paraffin removal, and interpreting a special stain without its control.
Worked example: A tissue section is PAS-positive. After diastase digestion, the positivity disappears, identifying glycogen. If magenta staining persists, the material is diastase-resistant-mucin or basement membrane-not glycogen.