H&E

Question 1

What is the difference between Hematin and hematein?

Answer 1

Hematein is the actual oxidized dye form of hematoxylin that stains nuclei

Question 2

What is the difference between progressive and regressive staining?

Answer 2

Progressive staining stains up to the desired intensity and then the reaction is stopped

Regressive staining intentionally over-stains the tissue and then differentiates for the desired element

Question 3

Steps in Progressive HandE

Answer 3

Xylene: removes paraffin
Alcohols: hydrate tissue
Tap water: removes alcohol
Hematoxylin (Mayer or acidified Harris): stains nuclei
Tap water: removes excess hematoxylin
Bluing agent: blues nuclei
Tap water: removes excess bluing
Eosin: stains cytoplasm
70% alcohol: dehydrates
95% alcohol: dehydrates
100% alcohol: dehydrates
Xylene: dehydrates
Coverslip: protects stained tissue

Question 4

Steps in Regressive HandE

Answer 4

Xylene: removes paraffin
Alcohols: hydrate tissue
Tap water: removes alcohol
Hematoxylin (Delafield, Ehrlich, or Harris without acid): stains nuclei
Tap water: removes excess hematoxylin
1% HCl in 70% Alcohol: differentiation
Running water: wash
Bluing agent: blues nuclei
Tap water: removes excess bluing
Eosin: stains cytoplasm
70% alcohol: dehydrates
95% alcohol: dehydrates
100% alcohol: dehydrates
Xylene: dehydrates
Coverslip: protects stained tissue

Question 5

What is a mordant?

Answer 5

Something that helps a dye bind to a tissue, usually a metal

Question 6

Mordant and use for Ehrlich

Answer 6

Aluminum, regressive

Question 7

Mordant and use for Delafield

Answer 7

Aluminum, regressive

Question 8

Mordant and use for Harris

Answer 8

aluminum, progressive unless acetic acid is absent from the protocol

Question 9

Mordant and use for Mayer

Answer 9

aluminum, progressive, no alcohol

Question 10

Mordant and use for Gill

Answer 10

aluminum, progressive, mucin

Question 11

Mordant and use for Weigert

Answer 11

Iron, not used for routine H&E, usually used in special stains

Question 12

2 Examples of acidic dyes

Answer 12

Orange G, picric acid

Question 13

2 Examples of basic dyes

Answer 13

crystal violet, safranin

Question 14

Purpose of bluing agents

Answer 14

Helps increase the intensity of the nuclear blue stain

Question 15

3 Examples of bluing agents

Answer 15

Lithium carbonate, ammonium hydroxide, or Scott solution (gentle alkaline)

Question 16

How does pH affect routine HandE staining?

Answer 16

If bluing agent is carried over to the eosin it will raise the pH and cytoplasm will be poorly stained

Question 17

What cells stain darkest with eosin?

Answer 17

Erythrocytes (RBC’s)

Question 18

Incomplete deparaffinization

Answer 18

Splotchy staining, with pale or unstained areas

Question 19

Nuclear staining is not crisp

Answer 19

“smudgy staining” when chromatin is not clear/crisp
commonly due to incomplete fixation
too much heat during processing or drying out of slides

Question 20

Pale nuclear staining

Answer 20

Presence of iron, sulfur, or chlorine in the water before or after hematoxylin
too short hematoxylin step
over-oxidized or depleted hematoxylin
over-differentiating the hematoxylin

Question 21

Dark nuclear staining

Answer 21

Highly alkaline or hard tap water
too long in hematoxylin
sections are too thick
differentiation was too short

Question 22

Red or red-brown nuclei

Answer 22

over ripened/old hematoxylin

or improper bluing step (you can’t over-blue)

Question 23

Pale cytoplasmic staining

Answer 23

pH of Eosin is too high, usually due to carryover of bluing agent
or over-differentiated in low concentration alcohols

Question 24

Dark cytoplasmic staining

Answer 24

May be due to alkaline or hard tap water
make sure not to over stain or under-differentiate
section thickness

Question 25

Eosin doesn’t have 3 shades

Answer 25

Improperly differentiated

Check pH of Eosin

Question 26

Blue black precipitate on sections

Answer 26

hematoxylin wasn’t filtered

Question 27

Hazy or milky water or slides

Answer 27

there is xylene in the water during dehydration

Question 28

Uneven HandE staining

Answer 28

incomplete deparaffinization or thick and thin sectioning

equipment malfunction

Question 29

poor contrast between nucleus and cytoplasm

Answer 29

Either of the stains is under or over-done

Question 30

Opaque slides

Answer 30

not properly deparaffinized
water in xylene causing cloudiness
water in hydrating alcohols (front end) over diluted

Question 31

Bead-like structures in the bottom of the 1st deparaffinizing Xylene

Answer 31

Water from the tissue if slides weren’t properly dried in the oven
moisture from the air
alcohol contamination in the xylene

Question 32

Washed out, dull nuclei

Answer 32

Overdecalcification
Over differentiated hematoxylin
Too short hematoxylin (progressive stain)
Too short bluing (you can’t over blue)
The hematoxylin itself is either over or under oxidized