I have trouble using Masson's trichome to view fibrosis in cardiac tissue. I simply followed the protocol I posted here from Sigma Aldrich. I let the frozen sections cool down to room temperature and then placed them in Bouin's solution overnight. I did not perform Step 1 nor the hydration step. I continued after Step 2 with the protocol. Also, these sections have not been fixed or paraffinized prior to freezing.
The main problem is that the Scarlet fades from the sections. It may not be binding. I attached two images for reference. I tried troubleshooting by increasing the time in each step. The Scarlet stained a little better, but pinkish white regions still appeared everywhere on the tissue. It improved a little when I washed off the picric acid using tap water, but the same results as the images appeared. Also the steps after Scarlet doesn't seem to make a difference, so I am thinking it may be the fixation steps.
I figure it might be due to picric acid and I read that some protocols use lithium carbonate and ethanol to remove it before doing the stain. Are there steps I need to do before fixing in Bouin's? I have not used this stain before and I am not sure what some of the terminology used in staining mean. Do I need to hydrate the slides before staining or is that for paraffinized slides only? Also, if I need to fix the slides before Bouin, is it possible to do that after the sections are frozen?
1. Deparaffinize slides and hydrate to deionized water.
2. Mordant in preheated Bouin’s Solution at 56°C for 15 minutes or at room temperature overnight.
3. Cool slides in tap water (18–26°C) contained in a Coplin jar for 5 minutes.
4. Wash in running tap water to remove ALL yellow color from sections for 1 minute.
5. Stain in Working Weigert’s Iron Hematoxylin Solution for 5 minutes.
6. Wash in running tap water for 5 minutes.
7. Rinse in deionized water.
8. Stain in Biebrich Scarlet-Acid Fucshin for 5 minutes.
9. Rinse in deionized water for 1 minute.
10. Place slides in Working Phosphotungstic/Phosphomolybdic Acid Solution for 5 minutes. Discard solution.
11. Place slides in Aniline Blue Solution for 5 minutes.
12. Place slides in Acetic Acid, 1%, for 2 minutes. Discard solution.
13. Rinse slides, dehydrate through alcohol (70%, 80%, 90%, 100%, 100%), clear in xylene for 5 minutes and mount.
Dear Judd,
I have only used Masson's trichrome on paraffin sections. However, this group has a protocol for processing mouse muscle frozen sections. Perhaps their modification will work for you. The do fix their frozen sections in 4% paraformaldehyde or 10% formalin before mordanting in Bouin's. Also note that they do not re-use the Beibrich Scarlet/Acid Fuchsin staining solution.
http://www.curecmd.org/wp-content/uploads/scientists/sop/MDC1A_M.1.2.003.pdf
If you are not locked into using Masson's, you may want to consider one of the one-step trichrome stains. They can be easier to use. A recipe for Gomorri's trichrome that works on frozen sections was posted here on RG:
https://www.researchgate.net/post/Any_advice_on_Massons_Trichrome_stain_on_frozen_muscle_sections
Good Luck!
Jill
Adjust the pH of the red dye to 2.0 with acetic acid (don't use hydrochloric acid!). Differentiate with phosphomolybdic or phosphotungstic acid just enough to bleach connective tissue (1 to 5 minutes or as per your tests). Stain with the blue dye pH adjusted to 2.0 with acetic acid (do not use hydrochloric acid!). Differentiate with 1% acetic acid for 1 to 2 min and pass in 100% alcohol once and xylene once. Mount with resinous mounting medium.
- Badges
- Science topic
- Similar topics
- Social Psychology
- Attitude