WangLab:Immostaining Extramembrane Signals

Immunostaining Protocol

 * 1) Prepare the cells
 * 2) *HeLa cells were transfected with membrane-targeted or cytosolic MT1-MMP biosensor.
 * 3) Wash cells twice with PBS.
 * 4) Fixation, staining with primary antibody, permeabilization and blocking
 * Primary antibody -> fixation ->   blocking
 * A) Chilled cells were incubated with GFP antibody (1:200 in CO2-independent medium without serum) at 4 C for 60 min.
 * B) Wash three times with ice-cold PBS to remove unbound antibody; Fix cells with 4% paraformaldehyde at RT for 20 min.
 * C) Wash three times with PBS.
 * D) Blocking with 10% BSA for 30 min.
 * Fixation -> Primary antibody -> blocking
 * A) Fix cells with 4% paraformaldehyde at RT for 10 min.
 * B) Wash three times with PBS; incubated with GFP antibody (1:200 in CO2-independent medium without serum) at 4 C for 60 min.
 * C) Wash three times with ice-cold PBS to remove unbound antibody
 * D) Blocking with 10% BSA for 30 min.
 * Fixation -> permeabilization -> Primary antibody > blocking
 * A) Fix cells with 4% paraformaldehyde at RT for 10 min.
 * B) Wash three times with PBS; permeabilize samples with 0.1% (v/v) Triton X-100 at RT for 20 min
 * C) Wash three times with PBS; blocking with 10% BSA for 30 min.
 * D) Incubated with GFP antibody (1:200 in CO2-independent medium without serum) at 4 C for 60 min.
 * E) Wash three times with ice-cold PBS to remove unbound antibody.
 * 1) Stained with fluorescence-conjugated secondary antibody (1:100) in 1% BSA-PBS at RM for 30-60 min.
 * 2) Remove secondary antibody; wash three times with PBS.
 * 3) Get ready mounting solution. Mount samples with antifade solution.
 * 4) Taking imaging under microscope.