Hi Jiwon, Here are a few considerations and potential troubleshooting steps: pH of Sirius Red solution: The pH of the Sirius Red staining solution is crucial for obtaining accurate and specific staining results. Typically, the pH is adjusted to acidic conditions (pH 1-3) to enhance the staining of collagen fibers. Ensure that the pH of your Sirius Red staining solution is within the appropriate range. Fixation: Proper fixation is essential to preserve tissue morphology and antigenicity. You mentioned using 4% paraformaldehyde (PFA) for 30 minutes, which is a common fixative. However, extended fixation times or inadequate tissue permeabilization can affect staining outcomes. Ensure that the fixation time is optimized for your specific tissue and experimental needs. Washing steps: Thorough washing steps after fixation and staining are crucial to remove any residual fixative or staining reagents that may interfere with the results. Ensure that you perform adequate and gentle washing steps with distilled water (DW) to remove excess staining and reagents. Acetic acid treatment: The acetic acid treatment step is typically performed to enhance the staining specificity of picro-sirius red for collagen fibers. However, excessive or insufficient acetic acid treatment can impact the staining results. Ensure that you perform the acetic acid treatment step according to the established protocol, as excessive dipping or insufficient acid treatment may lead to inconsistent staining outcomes. Optimizing staining conditions: If you are consistently observing red signals in the intratubular site unrelated to fibrosis, it may be beneficial to optimize the staining conditions. This can include adjusting the pH of the staining solution, incubation times, or concentrations of the staining reagents. Experimenting with different conditions and performing controls (such as omitting the staining reagents) can help identify the source of the unwanted signals.

To. Saman Behboodi Tanourlouee

Thank you to your answer!

I did additial experiments according to your advices.

I did change fixation time (30min, 1H and 2H) and pH (pH 1, 1.5, 2) of sirius red staining solution.

But it dosen't make a change... I got same results with upper picture...

I don't know how should I do and what it is problem.

I heared that before harvest kidney, reseachers should do reperfution.

I didn't reperfusion when I harvest samples.

It would be make problem?

Jiwon Jo

No I don't think so. If your tissues are paraffin-embedded, ensure proper deparaffinization and rehydration before staining.