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MLSCI250 HT LAB > LAB 3 Staining & Coverslipping > Flashcards

LAB 3 Staining & Coverslipping Flashcards

1
Q

Principle of Hematoxylin and Eosin stain

A
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2
Q

Principle of Periodic Acid Schiff

A
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3
Q

Principle of Masson Trichrome stain

A
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4
Q

Principe of de-paraffinization

A
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5
Q

Describe coverslipping

A
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6
Q

What is the difference between regressive and progressive staining ?

A
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7
Q

Common sources of error with each staining method

A
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8
Q

How are slides washed ?

A

Place slides under GENTLY running tap water for at least ONE MINUTE

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9
Q

How are slides rinsed ?

A

Place slides in tap water and gently change water 4 to 5 times

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10
Q

Describe the general staining process

A
  • done to completely remove all paraffin by soaking slides in xylene
  • place all slides to be stained in grey slide holder
  • slide is brought to the same solvent as the dye (ie. “bringing the slide to water)
  • after staining, slide is brought to a solvent compatible to the coverslipping agent
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11
Q

Although the post-staining treatment is the same as de-paraffinization, what makes them different ?

A

De-paraffinization: to completely remove paraffin

Post-staining treatment: to quickly get tissues to a hydrophobic state (ie. “trapping” ironically bound dye)

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12
Q

List process of de-paraffinization and post-staining treatment

A

De-paraffinization: Xyene, xylene, 100% alcohol, 100% alcohol, 95% alcohol, 80% alcohol, water (holding station)

Post-staining treatment: Water, 95% alcohol, 100% alcohol, 100% alcohol, xylene, xylene

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13
Q

List procedure for Hematoxylin & Eosin stain

A
  1. Wash for 1 min
  2. Harris’ Hematoxylin for 5 min
  3. Rinse
  4. 1 to 2 quick dips in acid alcohol (differentiation)
  5. Rinse
  6. 1% Lithium Carbonate for 30 sec
  7. Microscope check
  8. Eosin for 4 min
  9. Post-staining treatment
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14
Q

What is differentiation ?

A

Selectively removing excess dye

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15
Q

List procedure for Periodic Acid Schiff

A
  1. Wash for 1 min
  2. 1% Periodic Acid for 4 min
  3. Rinse
  4. Schiff’s Reagent for 15 min
  5. Wash for 5 min
  6. Harris’ Hematoxylin for 20 sec
  7. Rinse
  8. Post-staining treatment
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16
Q

List procedure for Masson Trichrome

A
  1. Iron (Weigert’s) Hematoxylin for 12 min
  2. Wash for 10 min
  3. 1% Acid Fuchsin/ Biebrich Scarlet for 5 min
  4. Rinse
  5. PMA/PTA
  6. Rinse
  7. Analine Blue for 5 min
  8. Rinse
  9. 1% Acetic Acid for 5 sec
  10. Rinse
  11. Post-staining treatment
17
Q

What should NOT be done to the mounting medium ?

A

DO NOT STIR mounting medium

18
Q

Do not stack newly stained slides for at least __ hours

A

Do not stack newly stained slides for at least 24 hours

19
Q

Hematoxylin: mechanism of action

A

Mechanism of Action:
- natural dye
- must be oxidized to hematein & mordanted before use
- regressive

20
Q

Hematoxylin: ideal appearance

A

Ideal Appearance:
- stains nuclei and basophilic substrates red at acidic pH and purple at basic pH
- over-stained (regressive)

21
Q

Acid Alcohol: mechanism of action

A
  • differentiation
  • removes excess dye
22
Q

Acid Alcohol: ideal appearance

A
  • dyes nuclei and basophilic substrates red at acidic pH and purple and basic pH
23
Q

Eosin: mechanism of action

A

Mechanism of Action:
- anionic dye with quinoid chromophore
- counterstain

24
Q

Eosin: ideal appearance

A

Ideal Appearance:
- counterstains basic components yellow and blue

25
Q

Periodic Acid: mechanism of action

A

Mechanism of Action:
- oxidation generates aldehydes

26
Q

Periodic Acid: ideal appearance

A

Pale purple

27
Q

Schiff’s Reagent: mechanism of action

A

Mechanism of Action:
- basic fuchsin treated with sulphuric acid
- pseudo-leuco dye
- must be washed excessively after

28
Q

What is a pseudo-leuco dye ?

A

A dye with two chemical forms affected by pH

29
Q

Schiff’s Reagent: ideal appearance

A

Ideal Appearance:
- red to magenta

30
Q

Harris’ Hematoxylin: mechanism of action

A

Mechanism of Action:
- counterstain

31
Q

Weigert’s (Iron) Hematoxylin: mechanism of action

A

Mechanism of Action:
- stains nuclei dark purple

32
Q

What is Bouin’s ?

A
  • combination of picric acid and formalin
  • stains tissue yellow
  • a post-fixation step in a staining method
33
Q

Acid Fuchsin/ Bebrich Scarlet: Mechanism of Action and Ideal Appearance

A

Mechanism of Action: displaces picric acid because Acid Fuchsin is larger
Ideal Appearance: stains everything red except for RBCs

34
Q

PMA/PTA: Mechanism of Action and Ideal Appearance

A

Mechanism of Action: displaces Acid Fuchsin because PMA/PTA is a larger molecule
Ideal Appearance: stains collagen pale yellow

PMA = phosphomolybdic
PTA = phosphotungstic acid

35
Q

Alanine Blue: Mechanism of Action and Ideal Appearance

A

Mechanism of Action: binds strongly to receptors on collagen
Ideal Appearance: stains collagen blue/ light green

36
Q

1% Acetic acid: Mechanism of Action and Ideal Appearance

A

Mechanism of Action:
- rinse
Ideal Appearance:
- brightens color

MLSCI250 HT LAB (5 decks)

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