I use the standard procedure here with frozen cardiac tissue and leave them in Bouin's solution overnight. I have problems with Scarlet which seems to fade. I tried troubleshooting by increasing the time in Iron Hematoxylin and Scarlet-Acid Fuchsin for 20 minutes and the Phosphotungstic/Phosphomolybdic Acid Solution for 10 minutes.
The Scarlet stained a little better, but faded pinkish white regions still appear everywhere on the tissue. It doesn't stain a strong red as a normal Masson's Trichrome.
The steps after Scarlet doesn't seem to make a difference, so I am thinking it may be the Bouin's solution. I use the standard procedure as shown below.
1. Mordant in preheated Bouin’s Solution at 56°C for 15 minutes or at room temperature
overnight.
2. Cool slides in tap water (18–26°C) contained in a Coplin jar.
3. Wash in running tap water to remove yellow color from sections.
4. Stain in Working Weigert’s Iron Hematoxylin Solution for 5 minutes.
5. Wash in running tap water for 5 minutes.
6. Rinse in deionized water.
7. Stain in Biebrich Scarlet-Acid Fucshin for 5 minutes.
8. Rinse in deionized water.
9. Place slides in Working Phosphotungstic/Phosphomolybdic Acid Solution for 5 minutes.
10. Place slides in Aniline Blue Solution for 5 minutes.
12. Place slides in Acetic Acid, 1%, for 2 minutes. Discard solution.
12. Rinse slides, dehydrate through alcohol, clear in xylene and mount.
[Edit] The Scarlet regions are faded in a pinkish-white color from the beginning. I do start with the Bouin's solution overnight in the standard procedure.
Do you see those white-pink regions in consecutive sections always at the same spot in the tissue or does it vary?
If they were at the same spot, it would show, that the tissue itself is the cause. Maybe a freezing artefact or something in the previous procedure? (necrotic area?)
When you say fading, do you mean, that the dye disappears after a while, or that the regions are not stained from the beginning?
Disappearing dye may be caused by residual water bevor coverslipping. This would be more look like "bleeding out" and is seen with aequous mounting medias.
The protocol is for paraffin-slides. Skip the first step for deparaffination and start directly with the Bouin. If you do this step with xylen and ethanols, the first contact of the unfixed tissue is with an organic solvent, then with ethanol, that would be the primary fixative and then with water. Nearly unfixed tissue may swell in water and loose soluble ingredients.
Do you see those white-pink regions in consecutive sections always at the same spot in the tissue or does it vary?
If they were at the same spot, it would show, that the tissue itself is the cause. Maybe a freezing artefact or something in the previous procedure? (necrotic area?)
When you say fading, do you mean, that the dye disappears after a while, or that the regions are not stained from the beginning?
Disappearing dye may be caused by residual water bevor coverslipping. This would be more look like "bleeding out" and is seen with aequous mounting medias.
The protocol is for paraffin-slides. Skip the first step for deparaffination and start directly with the Bouin. If you do this step with xylen and ethanols, the first contact of the unfixed tissue is with an organic solvent, then with ethanol, that would be the primary fixative and then with water. Nearly unfixed tissue may swell in water and loose soluble ingredients.
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