Alcian Blue - H&E Metanil Yellow Stain

Alcian Blue - H&E Metanil Yellow Stain

Reagents for this procedure are sold as individual stain solutions and are available for purchase under separate part numbers with storage requirements and expiration date designated per bottle.

Product Options:

Part # Non-Kit

 

SOLUTIONS: 250 ml 500 ml  1 Liter 1 Gallon
Acetic Acid 3%, Aqueous Part 10017A Part 10017B    
Alcian Blue Stain 1%, pH 2.5 Aqueous  Part 1003A Part 1003B    
Hematoxylin Stain, Mayer Modified   Part 1202A Part 1202B Part 1202C
Scott Tap Water Substitute   Part 1380A  Part 1380B Part 1380C
Eosin Y Working Solution   Part 1072A Part 1072B Part 1072C
Metanil Yellow Stain, Aqueous Part 12235B Part 12235C    

Additionally Needed:

Alcian Blue pH 2.5, Barrett’s Esophagus Control Slides Part 4023
Xylene, ACS Part 1445
Alcohol, Ethyl Denatured, 100% Part 10841
Alcohol, Ethyl Denatured, 95% Part 10842
Alcohol, Ethyl Denatured, 70% Part 10844

For storage requirements and expiration date refer to individual product labels.

 

APPLICATION:

Newcomer Supply Alcian Blue-H&E-Metanil Yellow Stain is a useful diagnostic screening method for Barrett’s esophagus and distinguishing from other gastrointestinal conditions/disorders.  The use of three separate stains and an eosin counterstain, provides a colorful combination of the gastrointestinal tracts various components.

 

METHOD:

Fixation:  Frormalin 10%, Phosphate Buffered (Part 1090)

Technique:  Paraffin Sections cut at 5 microns

Solutions:  All solutions are manufactured by Newcomer Supply, Inc.

All Newcomer Supply stain procedures are designed to be used with Coplin jars filled to 40 ml following the staining procedure provided below. 

 

STAINING PROCEDURE:

  1. Deparaffinize sections thoroughly in three changes of xylene, 3 minutes each.  Hydrate through two changes each of 100% and 95% ethyl alcohols, 10 dips each.  Wash well with distilled water.
  1. See Procedure Notes #1 and #2.
  1. Place slides in Acetic Acid 3%, Aqueous for 3 minutes.
  2. Move slides directly into Alcian Blue Stain 1%, pH 2.5 Aqueous for 15 minutes.
  3. Wash in running tap water for 5 minutes; rinse in distilled water.
  4. Stain in Hematoxylin Stain, Mayer Modified for 2 to 4 minutes.
  5. Rinse in running tap water until water is clear.
  6. Blue in Scott Tap Water Substitute; 2-3 dips.
  7. Wash well in running tap water; rinse in distilled water.  
  8. Place in 70% ethyl alcohol for 1 minute.
  9. Stain with Eosin Y Working Solution for 1 minute.
  10. Dehydrate in 95% ethyl alcohol for 30 seconds.
  11. Dehydrate in two changes of 100% ethyl alcohol; 30 seconds each.
  12. Place in Metanil Yellow Stain, Aqueous for 1 minute.
  1. See Procedure Note #3.
  1. Rinse/dehydrate in two changes of 100% ethyl alcohol. Clear in three changes of xylene, 10 dips each; coverslip with compatible mounting medium.

 

RESULTS:

Nuclei Blue
Cytoplasm Pink-Red
Mucin/ Barrett’s esophagus goblet cells Turquoise
Gastric mucin Some stain faint blue
Collagen Yellow
Smooth Muscle Salmon

 

PROCEDURE NOTES:

  1. Drain staining rack/slides after each step to prevent solution carry over.
  2. Do not allow sections to dry out at any point during staining procedure.
  3. If over-stained in Metanil Yellow Stain, Aqueous increased background staining will occur.
  4. If using a xylene substitute, closely follow the manufacturer’s recommendations for deparaffinization and clearing steps.

 

REFERENCES:

  1. Edgett, Wendi. Alcian Blue-H&E-Metanil Yellow Stain for Diagnosing Barrett’s Esophagus. Histologic 37.2 (2004). 35-38.
  2. Staples, Terri. “Alcian Blue-H&E-Saffron Stain for Gastrointestinal Biopsies”. Histologic 26.1 (1996).  2-3.
  3. Modifications developed by Newcomer Supply Laboratory.