Caspase immunofluorescence staining protocol
Procedure for detection of caspases using immunofluorescence.
Last edited Wed 14 Oct 2015
Apoptosis is a highly regulated mechanism of cell death, which converges on caspase activation. Immunofluorescence is a useful technique to detect caspases and other apoptosis-related proteins simultaneously in a cell sample.
This protocol should be used as a guide. Optimization will be required depending on the sample and antibodies used. Our antibody datasheets provide suggested working concentrations that should be tested in your own experiments.
Stage 1 - General procedure
Materials required
- Primary antibody against caspase eg Active Caspase 3 antibody, rabbit pAb (ab2302)
- Prepared, fixed samples on slides
- Triton X-100 or NP-40
- PBS
- Blocking buffer (PBS/0.1% Tween 20 + 5% appropriate serum)
- Conjugated secondary antibody eg goat anti-rabbit Alexa Fluor® 488 conjugate (ab150077)
- Mounting medium
- Humidified chamber
Steps
Permeabilize the fixed samples by incubating in PBS/0.1% Triton X-100 (0.1% NP-40 can be used instead) for 5 min at room temperature.
Wash three times in PBS, for 5 min at room temperature.
Drain the slide and add 200 μL of blocking buffer (PBS/0.1% Tween 20 + 5% appropriate serum).
- Lay the slides flat in a humidified chamber and incubate for 1-2 hours at room temperature.
- Rince once in PBS.
Add 100 μL of the primary antibody diluted 1:200 in blocking buffer.
- Incubate slides in a humidified chamber overnight at 4°C.
The following day, wash the slides three times, 10 min each in PBS/0.1% Tween 20 at room temperature.
Drain slides and add 100 μL of appropriate secondary conjugated antibody diluted 1:500 in PBS.
- Lay the slides flat in a humidified chamber, protected from light, and incubate for 1-2 hr at room temperature.
- Wash three times in PBS/0.1% Tween 20 for 5 min, protected from light.
Drain the liquid, mount the slides in a permanent or aqueous mounting medium and observe with a fluorescence microscope.