I found that Methanol fixation is not good for many of my antigens. Methanol has extremely negative impact on many of antigenic epitopes. I would suggest you using 3% PFA in PBS instead. Follow this protocol for fixation.

Fixation and permeablization:

• Add 3% PFA, and incubate for 20 at room temperature
• Wash 3 x 5 min with PBS
• inactivate residual PFA with 50 mM NH4Cl/PBS for 10 min (residual PFA can fix the antibodies used for staining)
• Wash 3 x 5 min with PBS
• Permeabilize cells with 0.1% TX-100 /PBS for 5 min
• Wash 3 x 5 min with PBS

and I hope you use proper blocking reagent. Goodluck.

Hi, I would go for the following protocol instead of fixation with Methanol. Start with treating coverslips with 1M HCl and rinsing/storing them dipped in sterile 1xPBS.

• Plate the cells at a density of 5K on each coverslip incubating inside cell culture dishes.
• Wash plates 3 x 5 min with PBS
• Fix in 4% PFA for 30min
• wash 2 x PBS
• Quench in 100mM Glycine in PBS for 15min RT
• Wash 2 x PBS
• Permeabilize in 0.1 Triton x100 for 8 min RT
• Wash 2 x PBS
• Block non specific Ab binding with 3% BSA in PBS for 30min
• Wash 2 x PBS
• Incubate in Primary ab for 1hr(diluted in 1:1000 PBS-BSA)
• Wash 3 x 5min PBS
• Incubate in secondary Ab diluted in PBS-BSA 1:200 for 1 hr RT
• Wash 3 x 10min PBS
• DAPI staining if required.

Thanks :)

How do you know that all your cells are infected? What time points are you harvesting your cells at? What MOI of infection are you using? At what confluence are you infecting? You might want to fix these before optimizing your transfection protocol.

Thank you all.

Just wondering did anyone tried tween 20 instead of Triton x 100?

Tween 20 is a mild detergent which can form large pores in the plasma membrane without dissolving the membrane, which is just good enough for antibody to pass. Refer the following article for more information.

Article Methods in Molecular Biology