Hello!
I'm staining mice retina with A488 and DAPI, the previous stainings worked well, but recent tissues turned white and almost no cell signal from both dyes despite the protocols and fixation are the same.
After mounting the tissue the successful ones look somehow halfway see-through, but the unsuccessful ones are more milky in color.
Thank you :)
Hi Enkhgerel,
one thing you might try, if not already done, is to perfusate the mouse: first with PBS at 37ºC in order to remove all blood from vessels and then with 4% pf, in order to pre-fix the retina. I have found that this helps keeping a better morfolgy...
Good luck!
It also sounds like water is trapped. I would reevaluate my mounting protocol. Clear tissue well before AHC.
Thank you both very much :)
1. Dr.Javier, for both specimen I use 4% PFA 0.1M PB perfusion fixation, and after enucleating the eyes, fix in the same fixator for 1 hour before dissecting the retina.
2. Dr. Michael A Kirby, will try the suggestion.
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