Immunostaining
This procedure was optimized using the 4G10 (anti-phosphotyrosine) antibody, but should work well for many other antibodies. For a generic protocol, add an additional 20-60min at RT fixing step after the 4C fixing step. New fixative is not needed, simply move the plate to the bench & cover with foil. Antibody concentrations will need to be optimized for each antibody. Please enter details for all optimized antibodies in the table at the end of the protocol.
Protocol
- Fix cells in 4% PFA/PBS for 20' at room temperature. To preserve membrane structures, fix with 4% PFA in PEM, 37C, 10 min.
- Wash 3X PBS
- Permeabilize and block using antibody buffer, 1hr at RT.
- Add primary antibody at desired concentration in antibody buffer, 20C for 1 hour or 4C overnight.
- Use hanging drop method:
- Put 100ul antibody mixture on a piece of parafilm
- Flip coverslip onto drop
- Cover with foil or box and leave undisturbed for 20 min (surface staining) or up to overnight (intracellular staining; minimum 1 hr). KEEP LEVEL.
- Use hanging drop method:
- Was 3X 15min with PBS.
- Add secondary antibody at desired concentration (typically 1:500 to 1:1000) in antibody buffer:
- Use hanging drop method:
- Cover with foil and leave undisturbed for 1-2 hours
- Cover with foil and leave undisturbed for 1-2 hours
- Use hanging drop method:
- Wash 3X 15min in PBS.
Cell can be imaged in PBS, or mounted using DAKO on a slide. Imaging is generally better in PBS, as DAKO can distort cells as it dries.
Recipes
PEM Buffer:
- 80 mM PIPES pH 6.8
- 5 mM EGTA
- 2 mM MgCl2
Antibody Buffer:
PBS + 0.1% triton X-100 + 5% donkey serum or 2.5% BSA (use goat serum of secondary is derived in goats).
For 10ml:
- 9.5ml PBS
- 10ul Triton X-100 (exclude if surface-staining)
- 500ul serum or 250mg BSA
Note: 1% BSA can be used in place of serum in many cases, especially after step 3.
Working Conditions
Antibody |
Fixation |
Primary Conditions |
Washes |
Secondary Conditions |
Washes |
4G10 |
4% PFA, 20', 4C |
1:100 in antibody buffer, overnight, 4C |
3 x 15min PBS |
1:200 anti-mouse in antibody buffer, 1 to 2 hours, room temp. |
3 x 15min PBS |
4G10 |
4% PFA, 20', 4C |
1:100 in antibody buffer, 4hrs, 20C |
3 x 15min PBS |
1:200 anti-mouse in antibody buffer, 1 to 2 hours, room temp. |
3 x 15min PBS |
THE His6 Antibody (GenScript A00186) |
4% PFA, 20', 4C |
1:250-1:500 in antibody buffer, 1-4hrs, 20C |
3 x 15min PBS |
1:1000 anti-mouse in antibody buffer, 1 to 2 hours, room temp. |
3 x 15min PBS |