Hello everyone!
I performed an ICC against cleaved caspase-3 in 4% pfa fixed cells (cultured with H2O2). I used an antibody that seems to work very well in ICC (from Cell Signaling), but my results weren't clear (attached photo)... Would be the problem the permeabilization step that I made with PBS + 0,1% Triton X-100 for 15 minutes? I used recomended antibody dilution and biotynilated secondary antibody conjugated to Cy3.
Hi Cl Pe
The issue here is not your ICC, but acquisition parameters on the microscopy.
The permeabilization method looks alright. I would try to optimize the microscope settings to get a brighter and sharper image. Its also a good practice to run secondary only controls, to get an idea of unspecific staining.
Hope this helps.
Best
Aparajita
You may have to recheck your permeabilization protocol and the extent of H2O2 exposure to the cells of your interest. You are targeting the cleaved Caspase-3 after H2O2 exposure, then it is beneficial to see the development of morphological apoptotic changes in the cultured cells. Once you are sure of getting those changes, then you can follow the ICC protocol with ensuring the permeabilization with Triton X-100. Several companies provide their own solution (for example R&D Systems provide the Cytonin) and give protocol for permeabilization of cells. I agree with your doubt, you may have to rule out the possibility. The second possibility is the H2O2 exposure itself since the overexposure can damage the intra-cellular caspase-3 due to its strong oxidizing as well as reducing properties. Hence make sure that you are using the recomonded H2O2 concentration and time of exposure. The third possibility is what Aparajita Lahree has suggested you that acquisition parameters on the microscopy.
In addition, kindly check the images of cells that were not exposed to H2O2 (control). Are you getting the similar image? Positive as well as negative control cells image using similar parameters of florescence microscope will be helpful to find out the exact cause of your problems. Good luck
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