hematoxylin and eosin stain Flashcards
how to de paraffinize tissue
bringing the slides to water, opposite of processing
hematoxylin
natural dye from logwood of H. campechianum tree
- it lacks a chromophore
- must be oxidized to hematein and mordanted before use
oxidizing occurs in 2 ways
- natural
- chemical
natural oxidation
exposure to air and light or UV
has a long shelf life
chemical oxidation
quick but can quickly over oxidize
- used immediately and discarded after
since hematein direct staining is weak what is used alongside
a mordant will enable attachment to tissue substrate
hematoxylins are classified according to mordant used: alum or iron
alum hematoxylin
most commonly used
used regressively
differentiation is usually acid alcohol
the alum-dye lake stain nuclei and basophilic substrates a reddish colour at acid pH, which is converted to a dark purple lake at pH 7-9
iron hematoxylin
dye mordant lake is darker and very resistant to differentation
eosin
anionic dye with quinoid chromophore
- different shades y=yellowish b=bluish
common counterstain to many staining methods
fixation used in H&E
most routine fixative are suitable
what is the result of a prolonged formalin
poor nuclear stain
what is the result of decalcification in H&E stain
decrease nuclear stain
mercury affect on H&E stain
increase eosin stain
what is the procedure for an H&E stain
- take slides to water
- overstain in alum hematoxylin
- wash in water
- differentiate with acid alcohol
- wash in water
- bluing- change the pH,
- wash in water and check microscopically - nuclei stain enough depth /colour
- counterstain with eosin
- dehydrate and coverslip
basophillic tissue components (purple to black)
-nuclei, ribosomal DNA, calcium deposits
