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@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
