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Lab

H&E Trouble Shooting

QuestionAnswer
White spots in section after deparaffinization Section not dried enough or not in xylene long enough; place in absolute alcohol then xylene or xylene longer
Nuclei too pale (hematoxylin light) Not in hematoxylin long enough, hematoxylin overoxidized, differentiation too long, decal'd tissues need longer; restain
Nuclei overstained (hematoxylin dark) Too long in hematoxylin, sections too thick, differentiation too short, Decolorize, restain or recut thinner section, check pH, rinse bluing cup, dehydrate quickly
Red, reddish-brown nuclei Hematoxylin old, hematoxylin not ripened, insufficient bluing, longer bluing (can't overblue), check hematoxylin
Pale staining w/ eosin Eosin pH >5 (maybe bluing carryover), sections too thin, dehydrated too long check eosin pH, don't let bluing carryover, check section thickness, don't let stand in alcohol, dehydrate quickly
Cytoplasm overstained, differentiation poor Eosin too concentrated (eosin-phloxine), section stained too long, not dehydrated enough, eosin in alcohols; dilute eosin, decrease stain time, longer dehydration, check section thickness
Blue-black precipitate on top of sections Overoxidzed hematoxylin; filter hematoxylin
Microscopic water bubbles in section Sections incompletely dehydrated; alcohol, xylene, recoverslip
Difficulty bringing some ares or tissue in focus w/ light microscope Mounting medium on coverslip; recoverslip
Mounting medium retracted from edge of coverslip Coverglass warped or mounting medium thinned w/ too much xylene; recoverslip, cap mounting medium
Water & slides turn milky following rehydrating alcohols Xylene incompletely removed; change alcohols, dehydrate slides
Slides hazy & milky in last xylene Water in xylene, change alcohol, redehydrate, change xylene
Mounted, stained sections don't show usual transparency & crispness under light microscope Mounting medium too thick; recoverslip
Hazy blue nuclei Too much heat on processor, too long in hot paraffin, too short fixation before dehydration; less heat, tissue should be well-fixed before processing, begin dehydration w/ 65% to 70%
Uneven H&E staining, nuclei show poor chromatin detail Water contaminated clearing agent, water or fixative contaminated infiltrating paraffin, solution levels insufficient; if open processor in high humidity use toluene instead of xylene, if closed processor check for malfunction
Dark basophilic staining of nuclei & cytoplasm, esp. around edges of tissue Laser & electrocautery heat artifact, can't fix
Brown stippling resembling pigment & section shows glossy black nuclei Section air-dried before coverslipping; remove coverslip, set in water several min, dehydrate, clear, remount
"Cornflaking" artifact caused by? Section drying before it is coverslipped
Poor contrast between nuclei & cytoplasm Understand nuclei, overstained cytoplasm; run control, check/change solutions
Created by: CCF