We´ve been using the Picro Sirius Red staining for several years now (done in out core facility) to show fibrosis development.
However suddenly our stainings, which usually had a yellow background turned orange, which makes the red fibers much harder to adequately quantify.
Our core facility hasn´t strayed from the usual protocol and no solutions were replaced or anything done differently than before, at least according to them.
We´ve done several stainings by hand, all of which have come out orange.
Does anyone have any idea how exactly this might have happened or how to make our background lighter yellowish as it was before?
How did you rinse the slides after picro sirius red staining? Did you rinse in acidified water? Was there any mistake preparing the acidified water?
Maybe prolonged rinsing clears the cytoplasma from the overstaining.
This would be my first trial.
Another would be to buy/make a new picro sirius red. The pH should be around 1-3. Maybe it has been contaminated with to much tapwater when carrying over the slides. The low pH is important to get a selective staining.
Gudrun Lang Thank you a lot for your answer!
I rinsed them under three different test concentrations of acetic acid (as recommended in different protocols).
They were 100% acetic acid, a 1:2 ratio and a 1:200 ratio of acidified dH2O.
There shouldn´t have been any mistakes when preparing the acidified water and we opened a new bottle of premade picro sirius red with a pH of 1.
Thanks again for your input, I´ll try prolonging the rinsing step and see where it gets me! :)
I believe It could be related to washing steps after staining or slides were not fixed with formalin. Check procedure (haematoxylin staining could be skiped) and comments here:
http://www.ihcworld.com/_protocols/special_stains/sirius_red.htm
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.
Please check my coments here for details:
https://www.researchgate.net/post/Picrosirius-overstaining-in-frozen-muscle
https://www.researchgate.net/post/sirius_red_staning
Daniil R. Petrenyov
Thank you too!
I found a solution by increasing the number of washes and instead of 100-80-40% EtOH went with three washes of 100 EtOH and a wash in pure acetic acid.
I will make sure to try your recommendations, too!
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