I am using the TCS-Bioscience kit for Masson trichrome stain of various tissues. I achieve good haematoxilin stain and red Ponceau Fuchsin for muscle tissue yet the light green Masson stain for collagen seems to dissipate to non-existence during dehydration and mounting.
Is there anything I can do to limit this or increase the intensity of the light green Masson collagen stain?
Thank you for any suggestions!
Protocol:
1. De-paraffinise slides - Xylene 3 min (x2), 100% IMS 3 min (x2) 70% IMS 3 min, 50% IMS 3 min, dH20 3 min (x2)
2. Incubate in Bouin's fixative overnight at room temperature.
3. Wash in running tap water.
4. Stain in freshly prepared Weigert iron Haematoxylin 5 min.
5. Wash in running tap water for 5 min.
6. Rinse in distilled or deionised water.
7. Stain with Ponceau Fuchsin (Masson) 5 min.
8. Rinse in distilled/deionised water.
9. Treat with Phosphotungstic/Phosphomolybdic Acid Solution 5 min.
10. Counterstain in Light Green (Masson) 5 min.
11. Immerse slides in 1% acetic acid solution.
12. Rinse slides in water
13. Dehydrate (See step 1 in reverse)
14. Clear and mount.
Look into Gomori's description. Replace by Mallory's Heidenhain and replace aniline blue by light green or fast green.
Or stain longer with fuchsin. Is your solution fresh, older one's do give problems.
It is the acetic acid step. Reduce the acetic acid strength and time to suit. Then rince thoroughly in freshly changed double-distilled (DD) water to remove any trace of the acetic acid. All washings in fresh DD water only.
Dear Samuel,
If you decide to try other forms of Masson's or Gomorri's trichrome stains, there are some other protocols at this RG thread:
https://www.researchgate.net/post/Any_advice_on_how_to_do_Massons_Trichrome_on_frozen_cardiac_tissue
Cheers!
Jill
i recommended It is best to cancel the acetic acid phase because it damages the dye and neutralizes it with distilled water
Thank you all for your answers. I tried multiple iterations, results as follows;
Reducing the acetic acid concentration and incubation improved the masson light green collagen stain slightly.
Entirely omitting this step caused no visible collagen stain at all.
Gomori trichrome gave far better results and was a quicker, more simple protocol - I will be using the Gomori stain in future.
Thank you again!
Sam
dear Samuel John Prendergast
at the protocol you mention at your question, you should change the solution of Phosphotungstic/Phosphomolybdic Acid Solution with aqueous Phosphotungastic acid at 5% for 15 minutes, this will give you a better result of light green, i have the same issue, i overcome this problem by this procedure's modification.
Apply this modification and tell me about the results, waiting it ?
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining