Immunocytochemical Analysis of Keloid Signaling

Immunocytochemistry was conducted as described previously [6 (link)]. Briefly, keloid fragments (5 mm × 5 mm × 5 mm) were fixed in 4% paraformaldehyde at 4°C for 48 h, embedded in paraffin, sectioned at 4 μm thickness by Rotary Paraffin Microtome Slicer paraffin embedding machine (Media Cybernetics, USA), and stained with hematoxylin and eosin for routine examination. Subsequently, the keloid sections were incubated with antibodies against p-PI3K (1 : 100, mouse anti-human PI3K monoclonal antibody, Santa Cruz, CA, USA), PTEN (1 : 100, mouse anti-human PTEN (a2b1) monoclonal antibody, Santa Cruz, CA, USA), p-Akt (1 : 100, mouse anti-human Akt monoclonal antibody, Santa Cruz, CA, USA), or p-mTOR (1 : 100, mouse anti-human mTOR monoclonal antibody, Santa Cruz, CA, USA). Bound antibodies were visualized using 3,3′-diaminobenzidine (DAB) as a chromogen (ZSGB Bio Co., Ltd, Beijing, China), and the slides were counterstained with hematoxylin. The positive expression of p-PI3K, PTEN, p-Akt, and p-mTOR was evaluated in four randomly selected fields under a light microscope (Olympus Corp., Tokyo, Japan). The mean optical densities (MOD) of positive expression were quantified with the Image Pro-Plus image analysis system (Media Cybernetics, Inc., Rockville, MD, USA).