Actually, i am trying the co-localize two proteins one of them is ER localize. The signals appear in the whole cell and difficult to localize. I am thinking a way to get rid of the cytoplasm from the cells before the start of the immunofluorescence staining. Is there a way to remove the cytoplasm from the cells on the coverslip, which remain intact and can be processed for the immunofluorescence.
Hello,
Indeed, sometimes it is necessary to perform a pre-extraction / post-extraction to better analyze the recruitment of protein using a confocal microscope. There are several different buffers that you can use depending on the material with which you work. You can triy with this one : Ice-cold PBS 1X containing 0.25% Triton X-100. If it does not work I'm sure you'll find plenty in the literature. Good luck!
Hi Ibrar
I would attempt some basic trouble-shooting steps before I started doing something radical like removing the cytoplasm.
Step 1: stain your cells with an ER tracking dying (as suggested by Sebastien above) - but only stain your cells with that dye. Can you see ER (and no cytoplasmic ) staining? If yes - move to step 2 - If no - you need to re-evaluate either your staining technique or the settings on the confocal.
Step 2: co stain for ER and your ER-specific protein (obviously using compatible dyes). If you still see cytoplasmic staining as well as ER staining then it is likely your protein is ubiquitously expressed throughout the cell.
all the best of luck to you!
- Badges
- Science topic
- Similar topics
- Engineering
- Petroleum Engineering
- Gas