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

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).

We recommend using serum from the host species of the secondary conjugate antibody (or closely related species) e.g. if using a goat anti-rabbit conjugate, use goat serum in the blocking buffer.​

Add 100 μL of the primary antibody diluted 1:200 in blocking buffer.

You can also prepare a slide with no primary antibody as a negative control.

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.

Drain the liquid, mount the slides in a permanent or aqueous mounting medium and observe with a fluorescence microscope.