Caspase immunofluorescence staining protocol
Procedure for detection of caspases using immunofluorescence.
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:
* 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.
- Permeabilize the fixed samples by incubating in PBS/0.1% Triton X-100 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 hr at room temperature. Rinse once in PBS.
- 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.
- 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.
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.
출처 : https://www.abcam.com/protocols/caspase-apoptosis-detection-protocol
Caspase에 대한 항체 살펴보기 Caspase staining kit 살펴보기
Caspase immunofluorescence staining protocol
Procedure for detection of caspases using immunofluorescence.
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:
Active Caspase 3 antibody, rabbit pAb (ab2302)
goat anti-rabbit Alexa Fluor® 488 conjugate (ab150077)
* 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.
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.
출처 : https://www.abcam.com/protocols/caspase-apoptosis-detection-protocol
Caspase에 대한 항체 살펴보기 Caspase staining kit 살펴보기