prepared by Roza Vazin

Purpose:

Hematoxylin is a basic stain that stains basophilic structures such as chromatin and ribosome’s a deep purple or blue. The advantage of this stain is that it provides a clear stain of the cell nuclei. Eosin is an acidic stain that stains acidophilic structures red. It provides a good stain for the cell membrane.

Helpful tips:

The sections used for H&E staining of Xenopus tropicalis tadpoles are made on a Vibratome Series 1000. The thinner the sections the more defined the staining. Forty microns is often a good thickness for the sections. The sections are placed on slides straight from the vibratome and set to dry on a slide warmer set at 40°C. When rinsing the slides, hold the slide at a 45° angle with the back of the slide facing the stream of water. This will help prevent the sections from washing off. It is helpful to bleach the embryos before sectioning to get rid of any pigmentation.

Reagents:

-Hematoxylin - Sigma Accustain Harris Hematoxylin (catalog # HHS-32)

-Acidic Alcohol (396 ml of 95% Ethanol, 4 mls concentrated HCl)

-Bluing Agent (1 liter water, 1 gm sodium bicarbonate)

-Eosin - Sigma Accustain Eosin Y (catalog # HT110-2-32)

-75%, 95%, 100% ethanol

-Histo-clear (order No. HS 200)

-Permount

H&E Stain

o Hematoxylin – 5 minutes

o Rinse in tap water – 30 seconds

-To remove excess stain.

o Acidic Alcohol – 5 dips

-To destain and differentiate.

o Rinse in tap water – 30 seconds

o Bluing Agent – 2 minutes

-This step is optional. The sodium bicarbonate changes the color of the hematoxylin-stained nuclei from purple to blue.

o Rinse in tap water – 30 seconds

o Eosin – 30 seconds

-The sections may be left in eosin for longer or shorter periods of time depending on the intensity of the stain desired.

o 75% Ethanol – 30 seconds

-Begins the stepwise dehydration.

o 95% Ethanol – 30 seconds

o 100 % Ethanol – 30 seconds

o Histo-clear – 10 seconds

o Place a few drops of permount onto

the slide before adding the coverslip.

Eosin Counterstain

-The previous protocol stains the tissues intensely and can be used to add contrast to sections. However, it may be desirable to use eosin as a counterstain to other stains. In that case, a much lighter stain may be desirable to allow the primary stain to predominate yet still allow visualization of other tissues.

o Eosin – 1 seconds

-Eosin stains very strongly so for a light background stain it is best to

stain for less than 5 seconds if possible.

o 75% Ethanol – 30 seconds

-Begins the stepwise dehydration.

o 95% Ethanol – 30 seconds

o 100 % Ethanol – 30 seconds

o Histo-clear – 5 seconds

o Place a few drops of permount onto

the slide before adding the coverslip.

H&E Protocol
prepared by Roza Vazin Purpose: Hematoxylin is a basic stain that stains basophilic structures such as chromatin and ribosome’s a deep purple or blue. The advantage of this stain is that it provides a clear stain of the cell nuclei. Eosin is an acidic stain that stains acidophilic structures red. It provides a good stain for the cell membrane. Helpful tips: The sections used for H&E staining of Xenopus tropicalis tadpoles are made on a Vibratome Series 1000. The thinner the sections the more defined the staining. Forty microns is often a good thickness for the sections. The sections are placed on slides straight from the vibratome and set to dry on a slide warmer set at 40°C. When rinsing the slides, hold the slide at a 45° angle with the back of the slide facing the stream of water. This will help prevent the sections from washing off. It is helpful to bleach the embryos before sectioning to get rid of any pigmentation. Reagents: -Hematoxylin - Sigma Accustain Harris Hematoxylin (catalog # HHS-32) -Acidic Alcohol (396 ml of 95% Ethanol, 4 mls concentrated HCl) -Bluing Agent (1 liter water, 1 gm sodium bicarbonate) -Eosin - Sigma Accustain Eosin Y (catalog # HT110-2-32) -75%, 95%, 100% ethanol -Histo-clear (order No. HS 200) -Permount H&E Stain o Hematoxylin – 5 minutes o Rinse in tap water – 30 seconds -To remove excess stain. o Acidic Alcohol – 5 dips -To destain and differentiate. o Rinse in tap water – 30 seconds o Bluing Agent – 2 minutes -This step is optional. The sodium bicarbonate changes the color of the hematoxylin-stained nuclei from purple to blue. o Rinse in tap water – 30 seconds o Eosin – 30 seconds -The sections may be left in eosin for longer or shorter periods of time depending on the intensity of the stain desired. o 75% Ethanol – 30 seconds -Begins the stepwise dehydration. o 95% Ethanol – 30 seconds o 100 % Ethanol – 30 seconds o Histo-clear – 10 seconds o Place a few drops of permount onto the slide before adding the coverslip. Eosin Counterstain -The previous protocol stains the tissues intensely and can be used to add contrast to sections. However, it may be desirable to use eosin as a counterstain to other stains. In that case, a much lighter stain may be desirable to allow the primary stain to predominate yet still allow visualization of other tissues. o Eosin – 1 seconds -Eosin stains very strongly so for a light background stain it is best to stain for less than 5 seconds if possible. o 75% Ethanol – 30 seconds -Begins the stepwise dehydration. o 95% Ethanol – 30 seconds o 100 % Ethanol – 30 seconds o Histo-clear – 5 seconds o Place a few drops of permount onto the slide before adding the coverslip.