[Histonet] Troubleshooting Gomori's Trichrome Stain (Blue Collagen, Richard-Allen) staining

[John Kiernan]

One-step trichrome methods (such as Gomori's) are OK when they work, but there's little you can do when the colours come out wrong. A simple thing to try would be a shorter time in the staining mixture, to reduce diffusion of the more slowly penetrating dye (aniline blue) into cells. Trichrome methods work better after coagulant fixation than after formaldehyde. (The Bouin pre-treament is to offset the undesirable effects of fixation in neutral formaldehyde; picric acid alone works just as well. See also Yu & Chapman 2003 J. histotechnol. 26(2): 131-134.)  If you can fix your hearts in Bouin or Carnoy you will get a better result with any trichrome technique.


If the one-step method still won't work, use a multi-step trichrome where you can have some control over the actions of the different components. Masson's (which has several variants) is popular; Mallory's has fewer steps. Good luck!


John Kiernan

Anatomy, University of Western Ontario

London, Canada

= = =

________________________________
From: abi jag via Histonet <histonet at lists.utsouthwestern.edu>
Sent: 25 July 2019 08:32
To: histonet at lists.utsouthwestern.edu <histonet at lists.utsouthwestern.edu>
Subject: [Histonet] Troubleshooting Gomori's Trichrome Stain (Blue Collagen, Richard-Allen) staining

Dear histo experts,Please provide me with your valuable suggestions for the problem described below.Objective: Staining rat hearts (fibrosis) with Gomori's one step Trichrome Stain (Blue Collagen, Richard-Allen) to demonstrate the collagenProcedure: Paraffin sections of 4 micron thickness; adequately fixed in 10 % NBF,  Bouin’s Fluid treatment at 56°C for 1 hour before staining. Follow the procedure exactly recommended by kit insert (please see below)Problem: Non specific diffuse bluish discoloration of cardio myocytes in the normal hearts, which looked completely odd. The staining of same section with picro sirius red came beautiful.Any insights on the potential reasons of this and ways to resolve?Thanks a lot in advance for your time and your vision to make a wealth of knowledge available in histonet.
Best regards,Abi

Standard Staining Protocol

1. Deparaffinize and hydrate sections to deionized water.

2. Place sections in Bouin’s Fluid at 56°C for 1 hour.

3. Rinse sections in running tap water for 3-5 minutes untilyellow color is removed.

4. Place sections in Working Weigert’s Iron Hematoxylin Stainfor 10 minutes.

5. Rinse sections in running tap water for 5-10 minutes.

6. Stain sections in Trichrome Stain for 15 minutes.

7. Place sections in 1% Acetic Acid Solution for 1 minute.

8. Rinse sections in deionized water for 30 seconds.

9. Dehydrate sections in 95% alcohol for 1 minute.

10. Dehydrate sections in two changes of anhydrous alcohol for 1minute each.

11. Clear sections in three changes of clearing reagent for 1minute each and mount.

_______________________________________________
Histonet mailing list
Histonet at lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Histonet Info Page - UT Southwestern Medical Center<http://lists.utsouthwestern.edu/mailman/listinfo/histonet>
lists.utsouthwestern.edu
Histonet -- For the exchange of information pertaining to histotechnology and related fields About Histonet