Can anyone help please... actually I wanted to probe membrane protein in MDA MB 231 cells so I stained the cells after fixing with 4% PFA, but antibody is staining inside the cells even without permeabilisation. I tried fixing cells in 2 % PFA but antibody is still staining inside. Have anyone faced such problem ? In inactive form this membrane protein reside as vesicles inside the cell. After staining the cells with respective antibody I could see the vesicles inside the cells even without permeabilisation.
Priyanka, I'm assuming from your question that your epitope is an extracellular epitope. If this is the case then I would recommend staining without fixation.
1. Cool your cells to 4'C by washing in ice-cold PBS. Make sure that your PBS is isosmotic with your growth media. Media is usually ~330-350mOsm.
2. You can block with 1mg/ml BSA in serum 30 min at 4'C
3. Add your antibody in BSA/PBS for 60 min at 4'C
4. Wash cells in BSA/PBS.
5. Fix cells in paraformaldehyde
6. Add secondary antibody as you normally would.
Hope this helps
Thanks Neil for replying. Actually I am labelling membrane protein that undergo recycling. In active form it is on membrane and is present as vesicle in inactive form inside the cell. If I will Block and Label cells at 4'C without fixing them I am afraid cells will undergo stress and their morphology will be disturbed that will again hamper my Imaging. What you suggest ?????
Your concerns are unfounded. This is exactly what my lab does all the time. We are looking at ion channels that traffick between the cell surface and endosomes and we image cells labeled at 4'C all the time. Don't worry, try it. Good luck
agreed with Neil, additionally paraformaldehyde works better at 4' C however fixation with formadehyde works better at RT or higher temp. thus alternatively you can try 10% formaldehyde for fixation surface labeling is better.
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