IHC tests the presence of a protein already expressed in the tissue, and 30 min are sufficient to allow recognition and binding of the antibody to the target protein. The secondary antibody is an HRP conjugated goat anti rabbit, that is a part of the Leica HRP-refine detection kit (Cat # DS9800). We have added the missing antibody information to section 2.10. We unfortunately do not have any tissue left to allow for performing WB analysis from the in vivo study, but we have conducted additional cell line studies and added WB analysis of cleaved PARP for the in vitro examinations and additional IHC examinations for autophagy and ER stress markers (as shown in response 3).
In the IHC experiment for PARP, the authors have incubated with primary antibody only for 30 min? Is that timing enough to get protein expression? What source of secondary antibody was used? In addition to IHC, I suggest performing western blot using PARP antibody where the full length and cleaved bands are observed in the same blot.