H&E Flashcards
What is the difference between Hematin and hematein?
Hematein is the actual oxidized dye form of hematoxylin that stains nuclei
What is the difference between progressive and regressive staining?
Progressive staining stains up to the desired intensity and then the reaction is stopped
Regressive staining intentionally over-stains the tissue and then differentiates for the desired element
Steps in Progressive HandE
Xylene: removes paraffin Alcohols: hydrate tissue Tap water: removes alcohol Hematoxylin (Mayer or acidified Harris): stains nuclei Tap water: removes excess hematoxylin Bluing agent: blues nuclei Tap water: removes excess bluing Eosin: stains cytoplasm 70% alcohol: dehydrates 95% alcohol: dehydrates 100% alcohol: dehydrates Xylene: dehydrates Coverslip: protects stained tissue
Steps in Regressive HandE
Xylene: removes paraffin Alcohols: hydrate tissue Tap water: removes alcohol Hematoxylin (Delafield, Ehrlich, or Harris without acid): stains nuclei Tap water: removes excess hematoxylin 1% HCl in 70% Alcohol: differentiation Running water: wash Bluing agent: blues nuclei Tap water: removes excess bluing Eosin: stains cytoplasm 70% alcohol: dehydrates 95% alcohol: dehydrates 100% alcohol: dehydrates Xylene: dehydrates Coverslip: protects stained tissue
What is a mordant?
Something that helps a dye bind to a tissue, usually a metal
Mordant and use for Ehrlich
Aluminum, regressive
Mordant and use for Delafield
Aluminum, regressive
Mordant and use for Harris
aluminum, progressive unless acetic acid is absent from the protocol
Mordant and use for Mayer
aluminum, progressive, no alcohol
Mordant and use for Gill
aluminum, progressive, mucin
Mordant and use for Weigert
Iron, not used for routine H&E, usually used in special stains
2 Examples of acidic dyes
Orange G, picric acid
2 Examples of basic dyes
crystal violet, safranin
Purpose of bluing agents
Helps increase the intensity of the nuclear blue stain
3 Examples of bluing agents
Lithium carbonate, ammonium hydroxide, or Scott solution (gentle alkaline)
How does pH affect routine HandE staining?
If bluing agent is carried over to the eosin it will raise the pH and cytoplasm will be poorly stained
What cells stain darkest with eosin?
Erythrocytes (RBC’s)
Incomplete deparaffinization
Splotchy staining, with pale or unstained areas
Nuclear staining is not crisp
“smudgy staining” when chromatin is not clear/crisp
commonly due to incomplete fixation
too much heat during processing or drying out of slides
Pale nuclear staining
Presence of iron, sulfur, or chlorine in the water before or after hematoxylin
too short hematoxylin step
over-oxidized or depleted hematoxylin
over-differentiating the hematoxylin
Dark nuclear staining
Highly alkaline or hard tap water
too long in hematoxylin
sections are too thick
differentiation was too short
Red or red-brown nuclei
over ripened/old hematoxylin
or improper bluing step (you can’t over-blue)
Pale cytoplasmic staining
pH of Eosin is too high, usually due to carryover of bluing agent
or over-differentiated in low concentration alcohols
Dark cytoplasmic staining
May be due to alkaline or hard tap water
make sure not to over stain or under-differentiate
section thickness
Eosin doesn’t have 3 shades
Improperly differentiated
Check pH of Eosin
Blue black precipitate on sections
hematoxylin wasn’t filtered
Hazy or milky water or slides
there is xylene in the water during dehydration
Uneven HandE staining
incomplete deparaffinization or thick and thin sectioning
equipment malfunction
poor contrast between nucleus and cytoplasm
Either of the stains is under or over-done
Opaque slides
not properly deparaffinized
water in xylene causing cloudiness
water in hydrating alcohols (front end) over diluted
Bead-like structures in the bottom of the 1st deparaffinizing Xylene
Water from the tissue if slides weren’t properly dried in the oven
moisture from the air
alcohol contamination in the xylene
Washed out, dull nuclei
Overdecalcification
Over differentiated hematoxylin
Too short hematoxylin (progressive stain)
Too short bluing (you can’t over blue)
The hematoxylin itself is either over or under oxidized
