DAPI staining in multiplex IHC is fading with stripping rounds?

29 March 2023 2 5K Report

Hello,

I currently do multiplex immunostaining on mouse small intestine swissroll sections. We apply the protocol described by Adrien et Guillot al (Article Deciphering the Immune Microenvironment on A Single Archival...

). Images are acquired on different days and the alignment of images is done via DAPI channel. We encounter the problem that DAPI staining is either fading with each antibody stripping round (although we restain for it), or that the nucleus itself is not stable anymore and desintegrating (please find attached an image). This problem is only seen with us and adjacent research groups in the university. the protocol works perfectly for other universities/clinics. We tried to compare what is different in terms of reagents, fixation time, dapi, slides etc, it seems everything is similar between us and others! anyone has an idea what the problem could be? without a stable DAPI staining we unfortunately cannot do multiplex on our samples.

Worth to note that we use a histology facility that process our sampels (and adjacent research groups) and do the alcohol dehydration paraffin embedding steps for us. We suspect that it could be that the processing time is too long and the tissue is loosing its integrity. We will try a shorter processing time soon. Thank you for your help and input.

Best,

Asmae

Anjali Srivastava

Hi Asmae Laouina

There could be several reasons for fading or unstable DAPI staining in multiplex IHC. Some possible causes and solutions are:

Overstripping: If the stripping buffer or conditions are too harsh, it can lead to loss of DAPI staining. Try reducing the stripping time, concentration of stripping buffer, or use a milder stripping buffer.

Photobleaching: DAPI staining is sensitive to photobleaching, especially if the slides are exposed to light for extended periods during imaging. Try minimizing exposure to light during imaging or use an anti-fade reagent to preserve DAPI fluorescence.

Tissue processing: As you mentioned, long processing times or harsh dehydration steps can lead to tissue damage and loss of DAPI staining. Try optimizing the processing protocol to minimize tissue damage and preserve nuclear integrity.

Antibody penetration: It is possible that the DAPI stain is not penetrating the tissue as well as the other antibodies, leading to inconsistent staining. Try adjusting the incubation time, temperature, or concentration of the DAPI stain.

Instrument or reagent variability: If the issue persists despite optimizing the above factors, it is possible that there is variability in the instrument or reagents being used. Try switching to a different DAPI stain or instrument to see if that improves the staining stability.

Overall, troubleshooting unstable DAPI staining requires a systematic approach to identify the underlying cause and optimize the protocol accordingly.

Asmae Laouina

Thank you very much for your input! we are checking the tissue processing step now. I will give a feedback if it works.

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