Hello dear community! :)

Just a quick update on my previous two posts.

After approximately 30 different protocol variations, I finally figured out why my staining wasn't working properly.

The problem turned out to be the very first staining step: Weigert's hematoxylin (working solution A:B = 1:1), which I had been incubating for 10 minutes. Once I omitted this step, both the Biebrich scarlet–acid fuchsin and aniline blue stained beautifully and produced the results I had been aiming for.

At the moment, I can no longer reliably visualize the nuclei, but for my current analyses that isn't essential. Thank you all so much for your suggestions and support throughout this troubleshooting process! 😊

If anyone has questions about what I tested, feel free to ask. I might also upload some pictures of the successful stainings tomorrow.

One additional observation: If I reduce the hematoxylin incubation to about 15 seconds, the nuclei become sufficiently visible in adult mouse cryosections, but the intensity of both the Biebrich scarlet–acid fuchsin and aniline blue decreases dramatically. Unfortunately, this shortened hematoxylin staining does not work for my embryonic chicken tissue.

Hi everyone,

Thanks again for all the helpful advice on my previous thread. Based on your suggestions, I started looking more closely at each individual staining step instead of only evaluating the final result.

One thing that stood out to me is that the tissue already seems to stain weakly during the Biebrich Scarlet step, before the PTA/PMA differentiation.

Several people suggested that I was probably over-differentiating, which is certainly possible. However, if the tissue never becomes intensely red after Biebrich Scarlet, then over-differentiation cannot be the only explanation.

Today's protocol was:

• Cryosections of adult mice hearts with induced infarcts (10 years old tissue, was stored at -20°C, results looked similiar to fresh embryonic chicken heart tissue stored at -80°C)
• No PFA pre-fixation
• Bouin's for 1 h at 35 °C
• Thorough washing
• Differentiation for 4 minutes in total and checking under microscope after every 2 minutes
• everything else according to the manufacturer's protocol

One thing I also noticed is that after the Bouin step the sections remain strongly yellow, even after 5-10 minutes of washing under gently running tap water. After 15 minutes in total they were clear though.

My questions are:

1. Immediately after Biebrich Scarlet, should the myocardium already appear intensely red before differentiation? If so, what could be the problem with my staining, as it appears to only accumulate the color in the rips and cracks of the tissue.
2. What could prevent Biebrich Scarlet from binding properly to the tissue?
3. Could prolonged retention of picric acid after Bouin interfere with the subsequent Biebrich Scarlet staining? Is this likely the case even after the tissue is not yellow anymore after the washing step?
4. Are there any cryosection-specific issues that could reduce Biebrich Scarlet uptake? One former colleague was performing successfull stainings following the protocol of the manufacturer.

Any thoughts would be greatly appreciated.

I attached some pictures made with Iphone on the ocular: before differentiation, after 2 minutes diff, after 4 minutes diff. But for me they all look the same. (40x magnification)

Before Diff

2 mins Diff

4 mins Diff

4x Magnification 4 min Diff

Hi everyone,

I'm currently trying to optimize Masson's Trichrome staining on cryosections of chicken embryo hearts, but the staining consistently turns out much paler than expected.

We are using the Polysciences Masson's Trichrome kit.

The myocardium stains only weakly pink, nuclei are poorly defined, and the contrast between myocardium and collagen is much lower than expected.

As a technical control, we also stained adult mouse heart cryosections in the same staining batch.

Current protocol

• Chicken embryo heart cryosections: 10–15 µm
• Adult mouse heart cryosections (technical control): 6 µm
• Sections stored at −80 °C for 3–5 days
• Thawed for ~5 min at room temperature
• 4% PFA fixation for 1 h
• PBS wash
• Bouin's mordant:
  • 1 h at 35 °C
  • OR overnight at room temperature
• Freshly prepared Weigert's Iron Hematoxylin 10 min
• Biebrich Scarlet 5min
• PTA/PMA differentiation 10min
• Aniline Blue 5min
• Acetic acid 2 min (1%)
• Standard dehydration and mounting (Ethanol 70%, 90%, 99%, Xylol)

All comparison slides were stained simultaneously using the same freshly prepared reagents.

What we've already tested

• Brand-new Polysciences kit
• Bouin's fixation for 1h 35°C incubation or for o/n RT

Despite this, both mouse and chicken sections remain much paler than previous stainings performed in our lab.

I've attached:

1. Previous successful mouse heart stainings (reference Pic 1+2)
2. Current chicken embryo heart stainings Pic 3
3. Current mouse heart stainings Pic 4

https://preview.redd.it/bhsej3x6wsdh1.png?width=960&format=png&auto=webp&s=657c0e74860b0b6bac2bb9ed5dab356cb54db388

https://preview.redd.it/zaonn2x6wsdh1.png?width=939&format=png&auto=webp&s=8aa91ea8129f690816be8ce7d436661ade12a679

https://preview.redd.it/p3lmf3x6wsdh1.png?width=754&format=png&auto=webp&s=2f12ae181be68b78ca85e537b03b1c1c2f9675e2

https://preview.redd.it/3jy1b4x6wsdh1.png?width=952&format=png&auto=webp&s=b3231ed7510ad8da900a48cbf79ef65626515221

Questions

1. Does anyone have experience with Masson's Trichrome on cryosections, especially embryonic tissue?
2. Could 1 h PFA fixation before Bouin already be excessive for cryosections?
3. Have you had to modify Biebrich Scarlet or PTA/PMA differentiation times for cryosections?
4. Are there any cryosection-specific modifications that made a significant difference for you?

Any advice would be greatly appreciated!