HIS H&E Staining & Coverslipping Flashcards
Color of Nuclei, cartilage, microorganisms in H&E
Blue-purple to black
Color of collagen and elastin in H&E
Pale pink
Color of cytoplasm in H&E
Shades of pink
Color of muscle fibres in H&E
Dark pink to red
Color of RBCs and Eosinophils in H&E
Bright red
Source of hematoxylin dye
- from heartwood of logwood tree (Haematoxylum campechianum)
T or F: Hematoxylin is a dye
FALSE; hematoxylin is NOT a dye—it is oxidized to form hematin, the dye responsible for its purple color
Natural oxidation
- uses light and air to oxidize hematoxylin to hematin
- 2-4 month process
- stable for years
Ie. Ehrlich’s and Delafield’s hematoxylin
Chemical oxidation
- oxidizes hematoxylin to hematin
- formed instantly
- shorter shelf-life; continues to oxidize hematin to colorless compounds
Ie. Gill’s, Harris’, and Mayer’s hematoxylin
Hematin requires a __ to stain predictably
Hematin requires a MORDANT to stain predictably
What compounds are used as a mordant for Hematin?
Aluminum or iron compounds
- also chrome, tungsten, molybdenum compounds
Why is hematoxylin used at a low pH ?
To suppress non-nuclear staining
NOTE: pH 1.0 - 3.0
Which mordant for hematoxylin is most routinely used ? Why ?
Potassium ALUMINUM sulfate or ammonium ALUMINUM sulfate; produce good nuclear staining, quick and easy to use
Describe staining with Alum Hematoxylin
- potassium aluminum sulfate OR ammonium aluminum sulfate
- stains nucleus brick red
- needs bluing (alkaline) solution = blue-purple nucleus
- used progressively or regressively
List bluing agents
- Lithium carbonate
- dilute ammonia
-Scott’s tap water substitute (magnesium sulfate, sodium bicarbonate, and water)
Regressive staining vs Progressive
Regressive: Over-staining the tissue, followed by differentiation with acid alcohol (removes stain from less desirable tissue elements)
Progressive: No differentiation; uses pre-determined time to stain nucleus without staining other tissue elements
List some factors that determine staining time
- age of solution
- formulation
- progressive vs regressive staining
- fresh/ frozen or fixed tissue
Disadvantage of Alum hematoxylin
Decolorized by acidic stain solutions = CANNOT be used for special staining techniques using acidified stains (Masson trichrome)
Iron hematoxylin uses ferric iron as both the __ and the __.
Iron hematoxylin uses ferric iron as both the MORDANT and the OXIDIZER.
Iron hematoxylin
- uses ferric iron as the mordant + oxidizer
- less stable; over-oxidized within a day = hematoxylin and iron are stored separately and mixed immediately before use
- mostly used progressively
- prolonged staining times
Most frequently used iron hematoxylin formulation
Weigert’s hematoxylin:
A= hematoxylin dye + 95% alcohol
B= aqueous ferric chloride + HCl + dH2O
Form of Eosin mainly used in histology
Eosin Y (yellowish)
Optimal pH for Eosin stain. What can be added to adjust pH ?
Acetic acid = (pH 4.5 - 5.0)
When properly differentiated, eosin alone can stain tissues at least __ shades of __.
When properly differentiated, eosin alone can stain tissues at least THREE shades of PINK to RED).
General staining procedure of H&E
- Bring slides to water (xylene>100%alcohol>95%alcohol>70%alcohol)
- Hematoxylin
- If regressive: differentiate w/ 0.5% acid alcohol
- Bluing w/ lithium carbonate
- Wash
- Eosin
- Dehydrate + clear: 70%alcohol>95%alcohol>100%alcohol>xylene)
- Coverslip
Cause+Troubleshoot: Hematoxylin is too pale
Cause: insufficient staining time, expired stain, over-differentiation, forgot to blue
Troubleshoot: increase staining time, use fresh stain, decrease time in acid alcohol
Cause+Troubleshoot: Hematoxylin too dark; cytoplasm is blueish
Cause: prolonged staining time, under-differentiation
Troubleshoot: decrease staining time, increase time in acid alcohol
Cause+Troubleshoot: Eosin is too pale
Cause:
- eosin contaminated with bluing reagent = increased pH decreases strength of anionic dye
- extended dehydration after eosin
- insufficient staining time
- expired eosin
Troubleshoot:
- check pH of eosin (4.6 - 5.0) = if >5.5, then replace eosin
- decrease time in dehydrating alcohols
- increase staining time
Cause+Troubleshoot: Eosin too dark
Cause:
- pH of eosin is too low
- prolonged eosin time
- insufficient dehydration time in alcohols
Troubleshoot:
- add NaOH to increase pH
- decrease time in eosin
- increase time in dehydration alcohols
Cause+Troubleshoot: Eosin does not have a 3-shade gradient
Cause: insufficient time in dehydrating alcohols, eosin pH is too high/low
Troubleshoot: increase dehydrating time in 70% alcohol, check eosin pH and alter as necessary
Cause+Troubleshoot: Blue-black amorphous artifacts
Cause: stain precipitate from Hematoxylin
Troubleshoot: filter stain before use (especially Harris)
Advantage of aqueous mounting media over resinous mounting media ?
Aqueous mounting media = can be used immediately after staining, without dehydrating/ clearing
Resinous mounting media = tissue must be dehydrated/ cleared prior to mounting
- crystal violet is alcohol-soluble, and fat in tissues will dissolve in xylene
Advantage of resinous mounting media over aqueous mounting media ?
Resinous mounting media = higher refractive index (1.5) = easier to focus on
- permanent, and stains will not fade
Aqueous mounting media= lower refractive index (1.4)
- NOT permanent, and stains can bleed
Cause+troubleshoot: Water droplets noted in stained sections after coverslipping
Cause: insufficient dehydration, clearing agent contaminated with water
Troubleshoot: remove coverslip by soaking in xylene, dehydrate and clear with fresh alcohol/ xylene before coverslipping
Cause+Troubleshoot: Some parts of the slide cannot be brought into focus after coverslipping
Cause: Mounting media on top of the coverslip
Troubleshoot: remove coverslip by soaking in xylene and re-mount with a new coverslip
Cause+Troubleshoot: corn-flaking artifact obscures section after coverslipping
Cause: section allowed to dry prior to coverslipping, mounting media was too thin
Troubleshoot: remove coverslip by soaking in xylene, re-hydrate section (soak in water for 15 min), dehydrate, clear, and re-mount
How to recognize water contamination of xylene ? How does this affect the quality of the slide ?
- xylene will be cloudy
- will not clear tissue = wax cannot infiltrate effectively during processing = water droplets on slide after staining
