Solutions:

3% Acetic Acid    Acetic acid    3.0 ml    Distilled water   97.0 ml    Stir together. Stable at room temperature for months.

Alcian Blue, pH 2.5    Alcian blue   1.0 g    3% acetic acid   100.0 ml    

Thymol crystals   Dissolve alcian blue in acetic acid. Check pH; adjust the pH as needed using acetic acid to pH 2.5.    Add a few crystals of thymol to prevent mold growth. Solution is stable at room temperature for    months and may be reused until weak.

Mayer Hematoxylin: Commercially made

0.25% Hydrochloric Acid     Hydrochloric acid     2.5 ml     Distilled water     997.5 ml     Carefully add hydrochloric acid to the distilled water slowly. Stable at room temperature for months.

0.25% Ammonia Water     Ammonium hydroxide     1.0 ml     Distilled water     250.0 ml     Slowly add ammonium hydroxide to distilled water. Use for one day only.

Eosin: Commercially made

0.25% Metanil Yellow     Metanil yellow     0.25 g     Distilled water     100.0 ml     Glacial acetic acid     0.25 ml     Mix together well. Stable at room temperature for up to one year.

Method:

1.   Deparaffinize and bring sections to water

2.   Stain with Alcian Blue, pH 2.5 solution15 minutes

3.   Wash well with water

4.   Stain in Mayer Hematoxylin *4 minutes

5.  Rinse in running water, several changes

6.   Differentiate in 0.25% hydrochloric acid 2-3 second

7.   Rinse in running water, several changes

8.   Blue in 0.25% ammonia water2 -3 seconds

9.  Rinse well in running water, several changes

10.   Place in 70% ethanol1 minute

11.  Stain with Eosin Solution1 minute

12.  Dehydrate in 95% ethanol30-60 seconds

13.  Dehydrate in 100% ethanol, two changes30 seconds each

14.  Place in Metanil Yellow solution**1 minute

15.  Rinse with ethanol, 2 changes10 dips each

16.  Clear with xylene, 3 changes2 minutes each

17.  Mount in a resinous medium

*For automated stainers, run a program on the stainer that takes the slides from water, through your routine H&E and stops at the second change of absolute ethanol.

** Timing of the Metanil Yellow is critical. If stained for too long, increased background staining will occur.

Results: Nuclei - blue Cytoplasm - pink-red Mucin – Turquoise for Barrett’s Esophagus Goblet Cells (some gastric mucin will stain a faint blue) Collagen - yellow Smooth muscle - salmon

Have fun :)

Following

Hello Amit,

You can find my H&E protocol and other options in the next question: https://www.researchgate.net/post/Does_anyone_have_detailed_protocol_for_H_E_staining

Alcian Blue protocol is this https://webpath.med.utah.edu/HISTHTML/MANUALS/ALCIAN.PDF

I use this protocol:

• Deparaffinize and hydrate to distilled water.
• 30 minutes AB
• Wash in running tap water, rinse in distilled.
• Hematoxylin, 3 minutes (according Hematoxylin type) (Or you can use Nuclear Fast Red).
• Wash in running tap water.

Also, you could try AB-PAS. I use this protocol:

• Deparaffinize and hydrate to distilled water.
• 30 minutes AB
• Wash in running tap water, rinse in distilled.
• Periodic acid, 10 minutes.
• Rinse in distilled water.
• Schiff's reagent, 3-6 minutes
• Wash in running tap water, rinse in distilled.
• Hematoxylin, 3 minutes, wash in water.
• Wash in running tap water.

You might adjust the times according the tissue and solutions.

You can find some protocols in these links:

• https://webpath.med.utah.edu/HISTHTML/MANUALS/MANUALS.html
• https://research.mcdb.ucla.edu/Lyons/Protocols.html

Good luck,

Juan