Do not use Mayer's Hx for trichrome stains. Best results are obtained using Weigert's Hx which has iron as mordant and is more resistant to removal under acidic conditions. Methyl blue is also a good alternative to using a green stain for collagen. The relative staining of muscle vs collagen is dependent upon the relative porosity of these structures when fixed and the relative size of dyes used. If both muscle and collagen are red, then slide requires more differentiation in PMA. This can take time to get right and you should check results every 1-2 minutes to see how things are progressing. Generally speaking, a brighter staining outcome is obtained when slides have been post fixed in picric acid for 30-60 minutes. I can supply full protocol if anyone is interested?

As you know Masson Trichrome is a good stain for distinguishing cellular from extracellular components.

Collagen fibers stain an intense green. Black or brown nuclei; mucus and ground substances take on varying shades of green. Cytoplasm stains red. Elastic fibrils, red blood cells and nucleoli stain pink.

Dear Mohamed

The original coloration of Masson has undergone numerous variations, the best of these is trichrome Masson mod. Goldner.

1) Stain sections dewaxed with hemalum acid Mayer

2) Wash in running water

3) stain for 5 minutiin:

Ponceau 2R 0.2 gr

Acid Fuchsin 0.1 gr

distilled water 300ml

acetic acid 0.6 ml

4) wash with a solution of 1% acetic acid

5) 5 minutes:

Phosphomolybdic acid 3-5 gr

Distilled water 100ml

Orange G

6) repeat step 4

7) Stain for 5 minutes:

Light green FCF 0.1-0.2 gr

Distilled water 100ml

Acetic acid 0.2 ml

8) Repeat step 4 for a long time

9) dehydrate in absolute ethanol and close the slide in synthetic resin

nuclei: blue black

cytoplasm; bright red to greenish

collagen fibers and acid mucopolysaccharides: Green decided

best regards

Fabio Nobili

sorry but I was wrong sender

is not Mohamed but is Nadia el Rouby.

Therefore ..... Dear Nadia el Rouby

thanks Dr. Fabio Nobili

Do not use Mayer's Hx for trichrome stains. Best results are obtained using Weigert's Hx which has iron as mordant and is more resistant to removal under acidic conditions. Methyl blue is also a good alternative to using a green stain for collagen. The relative staining of muscle vs collagen is dependent upon the relative porosity of these structures when fixed and the relative size of dyes used. If both muscle and collagen are red, then slide requires more differentiation in PMA. This can take time to get right and you should check results every 1-2 minutes to see how things are progressing. Generally speaking, a brighter staining outcome is obtained when slides have been post fixed in picric acid for 30-60 minutes. I can supply full protocol if anyone is interested?

Dear Dr. Harkin, Could I still use Mayer's Hx as an alternative to Weigert's A and cobine it with  Weigert's B (ferric chloride) for Trichrom staining?!

Regards

Hi all, I am attempting Masson's stain on formalin fixed, paraffin embedded human ileal strictures. I am having difficulty differentiating between collagen and muscle- both are staining red.

I start with Bouin's solution @ 56C for 1 hour, then run the stains. I initially differentiated in phosphomolybdic & phosphotungstic acid solution for 15 minutes, but  on examining the slides after this the collagen was still red. I left it in and kept checking, at 30 min it was still red.

The slides are 5 um thick, but I am planning to cut them thicker in future for other reasons.

How long is too long to leave in phosphomolybdic/tungstic acid? Any thoughts to help me?

Thanks!