Caspase immunofluorescence staining protocol

​​​​Procedure for detection of caspases using immunofluorescence.

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​​Introduction

Apoptosis is a highly regulated mechanism of cell death, which converges on caspase activation. Immunofluorescence is a useful technique when you want to detect caspases and other apoptosis-related proteins simultaneously in a cell sample.

Caspase immunofluorescence protocol

Materials required:​​

• Primary antibody against caspase e.g. Active Caspase 3 antibody, rabbit pAb (ab2302)
• Prepared, fixed samples on slides
• Triton X-100
• PBS
• Blocking buffer (PBS/0.1% Tween 20 + 5% appropriate* serum)
• Conjugated secondary antibody e.g. goat anti-rabbit Alexa Fluor® 488 conjugate (ab150077)
• Mounting medium
• Humidified chamber

​​*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.​

1. Permeabilize the fixed samples by incubating in PBS/0.1% Triton X-100 for 5 min at room temperature.
2. Wash three times in PBS, for 5 min at room temperature.
3. 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 hr at room temperature. Rinse once in PBS.                                             
4. 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. Incubate slides in a humidified chamber overnight at 4°C.
5. The following day, wash the slides three times, 10 min each in PBS/0.1% Tween 20 at room temperature.
6. 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.
7. Drain the liquid, mount the slides in a permanent or aqueous mounting medium and observe with a fluorescence microscope.

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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 which should be tested in your own experiments. Our range of caspase antibodies can be found here. 

We also have a range of  caspase staining kits for a simple and way of measuring active caspase in live cells.  An optimized protocol is provided with each kit, and should be followed carefully.