Immunohistochemical staining kit

Paraffin sections (2 μm in thickness) were used for all staining. Briefly, following blocking endogenous peroxidase with PBS containing 3% hydrogen peroxide for 5 min, sections were stained with primary antibodies for neutrophils (myeloperoxidase, MPO, Cat#: bs-4943R, 1:200, Bioss, Boston, MA, USA), macrophages (F4/80, Cat#: 28463-1-AP, 1:1000, Proteintech, Wuhan, Hubei, China), intercellular adhesion molecules-1 (ICAM-1, Cat#: 10020-1-AP, 1:1000, Proteintech), vascular adhesion molecule-1 (VCAM-1, Cat#: BM4289, 1:1000, BOSTER, Wuhan, Hubei, China), and P-gp (Cat#: 13978S, 1:1000, Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. Then, sections were incubated with Immunohistochemical staining kit (DAKO, Carpinteria, CA, USA) for 1 h at room temperature. All stained sections were counterstained with Giles’ hematoxylin and imaged on a fluorescent inverted microscope (Ts2R, Nikon). To quantify P-gp expression levels, neutrophil and macrophages, randomly selected fields from the ischemic cortex were analyzed by using ImageJ software.

IHC, co-immunoprecipitation and Western blotting were carried out, as described [12 (link)–15 (link), 17 (link)]. Antibody against WWOX/WOX1 was used for co-immunoprecipitation [12 (link)–15 (link), 17 (link), 22 (link)]. For IHC, skin tissue sections from patient were immersed in blocking solution (2% BSA) for 1 hr, followed by adding an aliquot of antibody against WWOX/WOX1 or TRAF2 for 2 hr incubation at room temperature. Following washing with PBS for 3 times, the tissue sections were incubated with aliquots of horseradish peroxidase (HRP)-conjugated secondary antibodies, and the resulting color was then developed using an immunohistochemical staining kit (Dako). In controls, no primary antibodies were used. The images were analyzed by an NIH Image J program.