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H&E Trouble-Shooting

Lab

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; restain
Nuclei overstained (hematoxylin dark) Too long in hematoxylin, sections too thick, differentiation too short, Decolorize, restain, check pH, rinse bluing cup, dehydrate quickly
Red, reddish-brown nuclei Hematoxylin old, hematoxylin not ripened, insufficient bluing (can't overblue)
Pale staining w/ eosin Eosin pH >5 (maybe bluing carryover), sections too thin, over-dehydrated
Cytoplasm overstained, differentiation poor Eosin too conc'd (eosin-phloxine), section stained too long, insufficient dehydration, eosin in alcohols
Blue-black precipitate on top of sections Overoxidzed hematoxylin; filter hematoxylin
Microscopic water bubbles in section Incomplete dehydration; 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 hot paraffin, under-fixed; begin dehydration w/ 65% to 70%
Uneven H&E staining, nuclei show poor chromatin detail Water in clearant, water or fixative in 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 Understained nuclei, overstained cytoplasm; run control, check/change solutions
Less than three shades of eosin Increase time in low dehydrating alcohols, best eosin differentiation occurs in 70%
Created by: CCF
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