WangLab:Immostaining Extramembrane Signals

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Immunostaining Protocol

  1. Prepare the cells
    • HeLa cells were transfected with membrane-targeted or cytosolic MT1-MMP biosensor.
  2. Wash cells twice with PBS.
  3. 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.
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