Abc alkaline phosphatase staining system

Standard histological paraformaldehyde fixation, paraffin embedding, and immunostaining were performed. Briefly, the skin biopsy samples were fixed in 4% paraformaldehyde for 24 h, and then embedded in paraffin. Sections of the skin (5 μm) were stained with hematoxylin-eosin (H&E) or toluidine blue to monitor the histological changes in the skin and recruitment of mast cell, respectively. Eosinophil peroxidase (EPX) staining was performed using a goat polyclonal anti–EPX antibody (Santa Cruz) and a ABC alkaline phosphatase staining system (Vector Laboratories) with 3,3′diaminobenzidine (DAB) as the staining substrate. NFκB/RelA staining within the skin tissue was performed using a rabbit monoclonal anti–NFκB/RelA antibody (Cell signaling technology) and Alexa Fluor 594 goat anti-rabbit antibody (Invitrogen). The nuclei within the tissues were counterstained using hematoxylin or 4′,6-diamidino-2-phenylindole (DAPI).

Immunostaining was performed using antibodies against cyclin A, cyclin E (Santa Cruz Biotechnology), with an ABC alkaline phosphatase staining system (Vector Laboratories) and 3,3′ diaminobenzidine (DAB) as the staining substrate. The nuclei of skin tissue were counterstained using hematoxylin.

Standard histological PFA fixation, paraffin embedding, and immunostaining protocols were performed. The skin biopsies were fixed in 4% (w/v) paraformaldehyde for 24 h, then embedded in paraffin. The sections (5 μm) were stained with H&E or toluidine blue to monitor histological changes of skin and mast cell recruitment, respectively. Eosinophil peroxidase (EPX) staining was carried out using a goat polyclonal anti–EPX antibody (Santa Cruz Biotechnology) and ABC alkaline phosphatase staining system (Vector Laboratories, Burlingame, CA, USA) with DAB as the staining substrate. The p65/RelA staining within the skin tissue was performed using a rabbit monoclonal anti-p65/RelA antibody (Cell Signaling Technology, Beverly, MA, USA) and an Alexa Fluor^® 594 goat anti-rabbit antibody (Invitrogen, Carlsbad, CA, USA). Cell nuclei within the tissues were counterstained using hematoxylin or DAPI.