Hello everybody,
I´m trying to stain frozen human muscle sections for collagen structures. For that I´m using Picrosirius Red Staining. The results are quite inconsistent with sometimes good staining results, where collagen structure between individual muscle fibers are stained red and muscle fiber are yellowish. Other sections will get completely red and it is impossible to distinguish between collagen and other structures. These inconsistent results also happen on the same slide of serial muscle sections using exactly the same protocol. See attached images from one slide.
Here is my protocol I´m currently using on frozen muscle cross sections:
- Samples out of -20 freezer
- Air-dry slides for 30 min room temperature
- Mark with DAKO pen
- Process the slides via 100-80-40 % EtOH
- 1 time 1 min MQ rinse
- Incubate the section for 30 min in (4%) formaldehyde
- 2 times 5 min MQ wash
- Incubate the section for 90 min picrosirius red (solution B Polysciences)
- 2 times 1 min 0.1N HCl solution (solution C Polysciences)
- 2 times 2 min MQ rinse
- 30s 70% EtOH
- 30s 95% EtOH
- 2min 100% EtOH
I already increased the time in ethanol at the end, but it didn´t improve the outcome.
Does anyone have an idea what might go wrong and how to change it? I´m looking forward to any kind of feedback.
Best,
Thorben
Dear, Thorben Aussieker .
The fixation in 4% formaldehyde is essential to minimize background staining. The processing of the slides via 100-80-40 % EtOH + 1 wash with water following 30 min in formaldehyde before PicroSirius Red staining helped me to get the right staining pattern.
Dmitry Zinovkin, so you would suggest to first fix with 4% formaldehyde, then rehydration steps and afterwards the PicroSirius Red staining?
Because so far I did: Rehydration, fixing, staining.
Matthew Ibbs yes, cut at the cryostat and then slides stored at -80°C and no chemical dehydration.
So protocol: water, formalin, stain with sirius red, acetic acid water, water, dehydrate?
Mohamed Mahdy solution A is Phosphomolybdic Acid and often used before the sirius red staining. But it didn´t show improvements in my first tries. Maybe I need to add it with the new suggestions.
Thanks already. I´ll give all options a try next week!
Dear all,
Cryosections could be stored frozen intect for ligand binding assays or dehydratated / delipidated with aceton. It could change staning as well.
In our case we found better results of Picro-Sirius red staning in slided processed for autoradiography and stored after dry at room tempreture in comparison to fresh sections. Probably repetative washing steps before staning improve results.
In my previous experience the fixation in formaldehyde is essential to minimize background staining in frozen sections. The processing of the slides via 100-80-40 % EtOH + 1 wash with water following 30 min in formaldehyde and wash before PicroSirius Red staining helped me to get the right staining pattern.
Protocol without haematoxylin staining adopted from
http://www.ihcworld.com/_protocols/special_stains/sirius_red.htm
Ready to use solution used from https://www.researchgate.net/deref/https%3A%2F%2Fwww.fishersci.com%2Fshop%2Fproducts%2Fsirius-red-1-in-picrid-acid%2F5030077
Please check my comments on link below for details with comparison of original and PFA fixed cryosections:
https://www.researchgate.net/post/Picrosirius-overstaining-in-frozen-muscle
https://www.researchgate.net/post/sirius_red_staning
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