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

1. Fix cells in 4% PFA/PBS for 20' at room temperature. To preserve membrane structures, fix with 4% PFA in PEM, 37C, 10 min.
2. Wash 3X PBS
3. Permeabilize and block using antibody buffer, 1hr at RT.
4. 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.
5. Was 3X 15min with PBS.
6. 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
       
7. 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