350 - Topic 9 (H&E and Coverslipping) Flashcards
the dye responsible for characteristic purple colour associated with hematoxylin
hematein
- via oxidation
natural oxidation of hematoxylin
light/sunlught and air to oxidize hematoxylin to hematein
- may take 2-4 mos
- solution will stay stable for years
- Ehrlich’s and Delafield’s
chemical oxidation of hematoxylin
uses sodium iodate (or another oxidizer)
- converts hematoxylin to hematein instantly
- can be used immediately
- but shorter shelf life
( maybe bc continue to oxidize beyond hematein to colourless compounds )
- Gill’s, Harris’, Mayer’s
what is a mordant?
a chemical that serves as a link between the dye and the substrate. The result is an insoluble compound that helps adhere the dye to the cells.
the choice of mordant will influence…
- which tissue components are stained
- what the resulting colour will be
T or F. All hematoxylin formulations are used at a high pH
F! low pH to suppress non-nuclear staining
which hematoxylin resists differentiation?
iron! strong covalent bonding
therefore only alum hematoxylins may be used progressively or regressively
most routine hematoxylin solutions are …
alum-mordanred
- potash or ammonium
- stains brick red then converted blue-purple by bluing agent
exmples of bluing agents
lithium carbonate
dilute ammonia
Scott’s tap water sub (mag sulfate, sod bicarb, water)
T or F. Alum hematoxylin may be used progressively or regressively
T!
Progressive vs. Regressive staining
Progressive: no diff step’ section stained for a pre-determined amount of time in order to stain nucleus without staining other tissue components
Regressive: over-staining; followed by diff step with acid alc which removes stain from less desirable tissue elements
what is stainig time depdent on ?
- age of solution
- formulation
- prog vs regressive
- whether tissue is fresh-frozen or fixed
one major disadvantage of alum hematoxylins:
they are decolourized by acidic staining solutions
so unsuitable for special staining techniques using acidified stains, such as MT
T or F. Hematoxylin is cationic
T! it is a basic dye; positively charged
stains neg charged tissue elements (nucleus)
most frequently used iron hematoxylin
Weigert’s hematoxylin
(sol’n A = hematoxylin dye + 95% alc solvent; sol’n B = aq ferric chloride mordant/ox, HCl acidifier, distileld water solvent)
why do we only combine sol’n A and B of Weigert’s hematoxylin immediately before use
it is less stable than alum mordanted hematoxylin
= can become over-oxidized within days so stored as two separate reagents
iron hematoxylns are not used routinely, but are frequently used in …
special staining (acidic stainig solutions spedcifically)
- MT
- VVG
an acidophilic xanthene dye
eosin
this may be added to eosin to control microbial growth
thymol
this is added to eosin to adjust pH
acetic acid; best staining = 4.5 -5.0
phloxine B
some labs use eosin AND phloxine B as counterstain
- more vivid shades of red
- other labs omit eosin and use phloxine B and saffron (HPS); this can differentiate collagen (yellow) from muscle + cytoplasm (shades of pink to red)
general staining procedure of H&E
- bring to water (hydrate) - remove paraffin via xylene, then absolute alcohol THEN hydrated and removing xylene through graduated alcohols and running water
- hematoxylin (5-45mins)
- water wash
- differentiate (OPTIONAL)
- bluing (DO NOT AGITATE)
- water wash
- eosin
- dehydrate, clear and mount - remove water by graduated alcohols then absolute alcohol(here the eosin is differentiated to different shades as well); xylene to remove alcohol and stay there until ready for moiunting
hematoxylin pale; poor contrast with eosin
- insuff. staining
- old stain
- over-diff
= forgot to blue (unlikely)
hematoxylin very dark; no chromatin details; cytoplasm bluish
- too long in solution
- under diff
eosin pale
- contaminated eosin with bluing agent
- extended dehydration after eosin
- old eosin (not likely)
- insuff staining time
what do we do when eosin is contaminated w bluing agent?
check pH and if above 5.5, change solution or re-acidify w acetic acid
eosin too dark; obscures cellular details
- pH of eosin too low
- too long in eosin
- insuff time in dilute alcohols during dehydration
less than 3 shades of eosin
- insuff time in dilute alcohols during dehydration
- pH too high or low
blue-black amorphous artifact noted overlying section
stain precipitate from hematoxylin => filter hematoxylin before use! Harris very prone to this
which hematoxylin demonstrates mucins
Gill’s stains goblet cells purple
which hematoxylins are slow and which are fast?
slow = Mayer’s
fast = Harris
gentle and alcohol-free hematoxylin
Mayer’s
can use for counterstain of oil red O b/c no alcs to dissolve lipids
routine mounting media
resinous
- transparent slides (easy to focus)
- once dry = permanent
what mounting media do we use if tissue elements can dissolve in alcohol or xylene
aqueous mounting media is used
- water-based
- don’t need dehydration or clearing
- simple syrups
- not as high of a refractive index as resinous mounting media (makes it difficult to focus on high-power)
- not permanent
- bleeding of stains can be a problem (can remedy by adding sugars; this can also raise refractive index slightly)
“corn flaking”
when slides dry before coverslipping
- disruptive brown artifact forms
- greatly reduces diagnostic potential
- nuclear drying artifact may also appear; obscures chromatin patterns
water droplets noted in stained section
- insuff dehydration
- clearing agent contaminated with water
some parts of slide cannot be brought into focus
mounting media on top of coverslip
cornflake artifact obscures the section
- section allowed to dry prior to coverslipping
- mounting media too thin
