Hematoxylin and Eosin Staining Method

Question 1

For routine microanatomical examination,_______is the staining method of choice

Answer 1

Hematoxylin and Eosin (H&E)

Question 2

H&E

The two stains were independently introduced in____ and ____, respectively, by____ and ____

Answer 2

1865 and 1875

Böhmer and Fischer

Question 3

In 1876,______ described their use in combination as a tissue staining method for staining different materials with different colours.

Answer 3

Wissowzky

Question 4

is the most important type of bonding that occurs in histologic staining techniques.

Answer 4

Ionic bonding

Question 5

It involves electrostatic attraction between ions of opposite charge, one of which is fixed in the tissue, and the second of which is in the dye

Answer 5

Ionic bonding

Question 6

alone is not technically a dye, and will not directly stain tissues.

Answer 6

Hematoxylin

Question 7

Hematoxylin needs to be used in combination with a “_____” – a compound that helps it link to the tissue

Answer 7

mordant

Question 8

The mordant used is typically a metal____, such as aluminium.

Answer 8

cation

Question 9

Hematoxylin in complex with aluminium salts is cationic and acts as a______ dye.

Answer 9

basic

Question 10

Hematoxylin is______ charged and can react with______ charged, basophilic cell components, such as nucleic acids in the nucleus.

These stain____ as a result.

Answer 10

positively

negatively

blue

Question 11

is anionic and acts as an acidic dye.

Answer 11

Eosin

Question 12

Eosin

It is_____ charged and can react with____ charged,_____ components in the tissue, such as amino groups in proteins in the cytoplasm.

These stain____ as a result.

Answer 12

negatively

positively

acidophilic

pink

Question 13

is a useful all-purpose stain that is also quick and easy to use. Despite its simplicity, this stain has stood the test of time.

Even now, over a century later, it remains the most frequently used tissue stain worldwide.

Answer 13

H&E

Question 14

Deparaffinization

It is the removal of wax is done with_____.

Answer 14

xylene

Question 15

It is essential to remove the wax completely, otherwise subsequent stages will not be possible.

At least _____changes in xylene are needed to ensure complete removal.

Answer 15

2 to 3

Question 16

Sections at this stage should appear clear and transparent.

Presence of any patches indicates the presence of wax and sections should be kept longer in the_____.

Answer 16

Deparaffinization

xylene

Question 17

_______is not miscible with aqueous solutions that are used later in the staining process.

Therefore it is removed with graded alcohol from higher concentration to lower concentration.

Answer 17

Hydration

Xylene

Question 18

Hydration

First change is made to____________ and finally_____.

Answer 18

absolute alcohol or acetone followed by 90, 70% alcohol

distilled water

Question 19

After hydration

Sections now should appear…

Answer 19

opaque

Question 20

Hydration

Presence of any clear areas are indicative of the presence of_____. To remove this, sections should be returned to absolute alcohol and rehydrated.

Answer 20

xylene

Question 21

(whenever necessary)

In case mercury containing fixatives e.g. Zenker is used, mercury pigments are precipitated on the sections.

Answer 21

Removal of mercury pigments

Question 22

Removal of mercury pigments

Removal involves treatment with____ solutions which changes mercury to an _____ compound.

This in turn is converted to______ by_____, which is readily soluble in water, the slides are placed in running water to wash out all extraneous chemicals.

Answer 22

iodine

tetrathionate

thiosulphate

Question 23

Staining Various staining reagents are applied during this hydrated stage.

This involves exposing the tissues to_____, _____ and strong chemicals like_____ and ______. Care must be observed during this stage to avoid detachment of tissues.

Answer 23

hematoxylin, eosin Y

acid alcohol and ammonia

Question 24

Dehydration and clearing

Dehydration is done using graded alcohols from_____ to _____

Answer 24

70% to absolute alcohol.

Question 25

can remove some of the stains so time has to be suitably modified to minimize fading

Answer 25

Dehydrating alcohol

Question 26

Since alcohol is miscible in xylene, it is used for clearing the sections.

Any sections from which water has not been completely removed would give a_____ appearance after the first xylene.

Such sections should be returned to _______and the process is repeated.

Mounting is done after ___ or ____xylene.

Answer 26

milky

absolute alcohol

2nd or 3rd

Question 27

From the slide warmer, allow the slide to cool for at least___ minutes. Then proceeding with staining

Answer 27

5

Question 28

Procedure

1. (2 changes, 5 minutes each).
2. (10 dips)

3.(3 changes, 10 dips each)

4. Wash slides in_____ and remove excess______ or impurities using a cotton swab. Remember not to touch the tissue sections. Then rinse the slide well with clean water and drain excess water on paper towels.
5. Stain slide in Hematoxylin for ______minutes. Drain excess stain then rinse with water.
6. Decolorize sections in_____ (2 quick dips only). Rinse slides immediately in water.

3|Page 7. Immerse slides in _____until tissue turns blue (average of 5 minutes). Rinse again in water. Drain excess water on paper towels.

8. Stain slide with Eosin Y for ____minutes. Drain excess stain then rinse well with water. Drain excess water on paper towels.
9. Dip in_____(3 changes, 10 dips each).
10. Lastly, dip slides in____ (3 changes, 10 dips each).

Answer 28

Xylene

Alcohol formol

95% ethanol

soapy water

albumin

5-10

acid alcohol

ammonia water

1.5 - 3

95% ethanol

xylene