I have seen crystal-like artifacts in ORO staining. those artifacts are also ORO positive. does anybody face this problem and has any solution please share.
I filtered the stain with a 0.22um filter after diluting the stoke solution in the DPBS.
After ORO stain washes several times.
Thank You
attachments: ORO stain images by brightfield/ confocal microscopy
Hi Mahesh,
It seems your staining of lipid have no issue and these crystal like artifacts are some kind of impurity during the processes. Could you please tell what protocol you are using to stain and what tissue are you trying to analyse.
thanks for the answer Anurag Nishad . I am trying to stain the adipocytes. procedure is. 30 minutes fix the cells with 4%PFA. then 5 minutes 60% isopropanol, then 20 minutes ORO, then 2 minutes 60% isopropanol, then 3-time DPBS washing, Then 30 minutes DAPI, 3 washing DPBS. and mount with wet mounting media.
ORO tends to precipitate if it is older than 6-7 months(Stock) no matter how many times you filter it. I suggest you to try staining with a new ORO stock stain. Maybe the artifacts will disappear.