H&E staining Flashcards
What are the three major steps in staining
Rehydration - take to water
-After dewaxation with xylol, you rehydrate with decreasing alcohol tap water (1 min) and then distilled water (1 min)
(removed wax with xylol and remove xylol with absolute)
(low grade alcohol is 0.70, 70% alcohol)
Staining - with reagents and stains
Dehydrate , Clear mount (permount) and Coverslip
dehy with increasing alcohol (0.95, abs, abs) and clear with xylol
water cannot mix with xylol (green bucket)- can cause improper rehydration and incomplete clearing
if you dont know where it was dip in 100% alc and itll be okay
What is the primary purpose of H&E stain
demonstrate nuclear and cytoplasmic staining
What does hematoxylin target
nuclear staining
blue or purple with crisp nuclear membrane surrounding the cells and dense purple mass inside - nucleolus
What does Eosin target
cytoplasmic staining - 3 shades of pink
collagen - light
muscle - dark
RBC - darkk pink/red
What makes hematin a true dye
hematoxylin has be oxidized to be a dye = hematin
1.Presence of chromophore groups- give color C, O, N with single or double bonds C=O, C=C
2.Presence of a strong chromophore – quinoid ring- with double bonds = intense color
3.Potential auxochromes- enable dye to link to tissue
What are the disadvantages of hematein
1.Inferior quality of hematein dye
2.Short shelf life of hematein dye
3.Hematein dye is expensive
What is a mordant
metallic mordant has a strong affinity to nuclei
mordant + dye = lake
how does oxidation of hematoxylin - ripening occur
1.Exposure to atmospheric oxygen
Delafield, Ehrlich solutions
- Sodium iodate, mercuric oxide, potassium permanganate
Harris, Mayer, Gill solutions
solutions should always have unoxidized hematoxy because oxidation continues with staining in air
how can you tell if the hematoxylin is fresh
color
Purple - fresh mordant
Red - aged but usable
Brown - over-oxidized
Metallic sheen on standing - aluminum hematein
Filter before use - blue-black precipitate on slide
how can you prevent scum formation on hematoxylin
Acetic acid - slows oxidation
what is a nuclear stain made up of
Mordant - aluminum or iron sulfate- dye to tissue
Oxidizer - sodium iodate, mercuric oxide
Stabilizer - glycerol
pH adjuster (Accentuator) - Citric acid - increases selectivity of the hematin to PO3 of nucleic acid by decreasing ion groups
oxidation is quicker in alkaline solution slower in acidic = increase shelf life
Prevents scum formation - Chloral hydrate or acetic acid
Solvent - Alcohol, water, ethylene glycol
Progressive VS Regressive
Progressive
Staining just long enough to reach proper endpoint
Less time (2 mins)
Regressive- dye with mordants
Overstaining and then destaining
More time (>5 minutes)
What is differentiation
-excess stain is removed by the regressive method
-uses 1% HCl in 70 % ethanol (acid alcohol)
-the H+ ions compete with mordant for tissue groups
-break the lake tissue link
-PO4 group of NA has a high affinity for lake so itll retain the color while others are colorless (cytoplasm)
results
Cytoplasm & connective tissue - colorless
Nuclei - red, with sharp nuclear detail
NEEDS TO BE REVERSED- counter stain is pink so you want contrast
Decolorization VS Differentiation
Decolorization - removal of excess stain macroscopically
Differentiation -controlled removal of excess stain microscopically
What is bluing
-reverses the color change that occurred during differentiation
-changes the solubility of the dye lake (aluminum-hematein is red and soluble in pH <5; blue lake is insoluble in pH >5)
weakly alkaline solutions:
Alkaline running tap water (RTW)
Lithium carbonate
Dilute ammonium hydroxide
Scott’s TWS
Cytoplasmin staining consists of
Eosin Y, Eosin B
Sodium-salt of a colored acid in powder form
negative charge from salt being lost from COO= auxochrome
What is eosin
Chromophore - in the anionic part of the molecule
@ pH 7 - negatively charged
@ pH < 6 [4.6 - 5] - can stain proteins (IEP pH 6)
@ pH < 4 - decreased negative charges on the dye because the molecule converts to a free acid
-Free acid may bind to tissues by hydrogen binding - “MUDDY tissues”
Results after cytoplasmic staining
Nuclei - blue
RBC - dark pink
Muscles - pink
Collagen - light pink
H&E technique
Harris Hematoxylin -Nuclear staining
Acid Alcohol-Differentiation
Scott’s tap water substitute -Blueing
Eosin Y-Cytoplasmic staining
Rehydration (“Take Down To water”)
Xylene – 5 min
Xylene – 2 min
100% EtOH - 1 min
95% EtOH - 1 min
70% EtOH - 1 min
RTW - 1 min or Slides can safely stay here
Manual progressive staining method
Harris hematoxylin 1-2 min
RTW 1 min
Scott’s tap water substitute 1 min
RTW 1 min
Eosin Y 20-30 sec
RTW 5 dips
DCM
no differentiation step
Manual regressive staining method
-Nuclear staining (Hematoxylin) 5 min
-RTW - 1 min
-Differentiation (Acid alcohol) - 1-2 quick dips
-RTW - 2 min
-Blueing (Scott’s tap water substitute) - 1 min
-RTW 1 min
-Check differentiation microscopically -
-Counterstain (Eosin) - 30 sec
-RTW 5 dips
-DCM
Dehydration, clear and mount (DCM)
95% EtOH - 5 dips
100% EtOH - 5 dips
100% EtOH - 5 dips
Xylol 1 min
Xylol Slides can safely stay here
Mountant
chromophore vs chromogen
chromophores can be reduced- color loss = chromogen = no color but can become a dye
