General notes on fixing and permeabilization for Immunoflourescence
1) See “Fixing and Immunofluorescence” protocol for general hints on IMF and fixing.
2 ) Depending on the antibody…. General fixatives are cold methanol, ethanol, acetone or no-fixation for fixation labile antigens; 1-2% paraformaldehyde in PBS for 10 minutes will do for many cases, a higher concentration of paraformaldehyde and lower concentrations (0.1%) of gluteraldehyde) for tough antigens and or labile structures.
2) 0.1% Triton X-100 in PBS for permealbilization usually 5 minutes but do a time course and diliution expt for opmizing.
3) Blocking with 5% goat serum and 1% BSA is the best block I have used, usually at RT or 37°C.
4) Use 1% BSA in PBS or goat serum for diluting antibodies.
5) Mounting media- Mowial (see my other protocols) Prolong antifade commercially available, DABCO (0.5g in 5mL glycerol, mixed in hot water bath to dissolve, then add 200mM Na2HPO4 to 10mL). Store in dark bottle or tubes.
1) Cannot tell which side is which? Place coverslips vertically in PBS, look at the surface of each side very obliquely. The cell side is white and cloudy, the other side is glossy.
2) No or low signal, try different fixations, lower dilution of antibody, different antibodies.
3) High background, gluteraldehyde may cause high background and autofluorescence, try higher dilution of abs, use affinity purified antibodies, less gluteraldehyde, blocking of free aldehydes with 5mg/mL NaBH4 in 1X PBS This blocks the free aldehyde groups When you make this be VERY CAREFUL. DO NOT mix the solid with the PBS near ANY source of FLAME. Make sure all bunsen burners are off or you could cause an explosion, leave the lid of the falcon tube ajar so that H bubbles can escape. Make this before you start since it needs to simmer down a bit before you use it.
4) Distorted structures: tougher fixation, do not let cells dry out.