AbcamCaspase immunofluorescence staining protocol

2020-12-01

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.


  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.


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

TOP