Hi,
My study involves tissue grinding of prostate tissue embedded in epoxy resin. Then performing Masson's trichrome staining for collagen on the exposed face of the grinded block face, but remains but the staining is very faint. Does anyone have any suggestions on how I can improve the staining?
The reagents for Masson's trichrome were purchased from Sigma:
- Weigerts Iron haematoxylin, equal parts of part A and B
- Biebrich Scarlet -acid Fuchsin solution
- Phosphomolybolic Phosphotungstic acid solution
- Aniline blue solution
- 1% acetic acid
The staining was performed at 70 deg C as follows:
- incubate in weigerts Iron haematoxylin for 2 minutes
- wash in water
- incubate in biebrich scarlet acid fuchsin solution for 3 min
- wash in water
- differentiate in Phosphomolybolic Phosphotungstic acid solution till collagen is pale pink ( about 12 minutes)
- rinse in water
- rinse in 1% acetic acid
- incubate in aniline blue solution for 1 minute
- rinse in 1% acetic acid 30 seconds
In the first image I have attached (1) shows red tissue with a blue stained collagen lining a vessel. However in the other image (3) other portions of the tissue show patchy blue staining. Also the resin picks up the red stains which obscures visualizing the tissue.
Does anyone have any comments on how optomised the staining is and / or suggestions on how to improve the staining?
thankyou
Ceara
I would suggest increasing the exposure to aniline blue to 7 minutes. This is a plastic section protocol that might help: https://www.urmc.rochester.edu/urmc-labs/pathology/stainsmanual/index.html?MASSONSTRICHROMESTAINFORGMAPLASTICBONEMARROWSECTIONS
Alternatively, have you tried Ladewig trichrome stain? http://stainsfile.info/stain/conektv/tri_ladewig.htm
The easiest answer I can give is that if you only need to visualize collagen you can omit the Biebrich Scarlet/Phosphomolybolic acid and only stain for collagen. There are also other stains for collagen (Van Geison) that may be visualized better. I just had to look this one up, but there is also a stain using PicoSirius Red that is visible under a polorized scope; I have not done that one myself but found a decent summery.
Typically prostate is not embedded in epoxy, which is reserved for harder structures that need to be visualized or for sections that need to be ultra-thin.
https://journals.sagepub.com/doi/10.1369/0022155414545787?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub++0pubmed&
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