My experiment involves a protein conjugated to a YFP tag inside HEK cells. When I add my treatment to the cells, the YFP is redistributed into punctate structures which seem to co-localize with autophagic markers (using IF).
When I went to replicate my IF experiment I found my puncta were no longer visible. It appears if I fix my cells (4% PFA, 15 min), permeabilize (0.1% Triton X-100, 15 min) and immediately mount onto slides the puncta remain, but they are reduced after 1h and disappear entirely after an overnight incubation. Has anyone experienced this or can suggest alternative protocols? Any help would be greatly appreciated. Thanks!
I have tried the following thus far with no luck:
- Fix, permeabilize and fix again
- Fix and permabilize with 100% methanol for 20 min
If your target is a vesicle, you should be thinking about its unstable. In my case, my target is surface protein on the lipid droplet. So I used the 0.05% Triton-X and 4% sucrose in TBS to do the permeabilization 5 minutes RT. (or you may be using the minus detergent, like the Digitonin and saponin)
Hi Maria, if your cells are intact then PFA will fix membrane proteins. when you add TX-100, all membranes will be dissolved.
I think Wei-Chun is right that using TX and PFA together might help.
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