Fig 1. Iba1, BDNF and P2X4R detection using immunocytochemistry (ICC).

ICC study to detect the expression of microglia-specific markers (Iba1, A) and proteins involved in the signaling pathway initiating pain hypersensitivity (P2X4R, B; and BDNF, C) in SIM-A9 cell line. SIM-A9 cells (Passage no. 9; P9) were cultured in a 96-well plate in complete growth media for 48 h. The cells were fixed with 4% PFA followed by immunostaining for Iba1, P2X4R, and BDNF. The cells were captured under phase contrast setting (Transmitted) and nuclei were counterstained using Hoechst 33342. The signals for P2X4R were imaged under the green fluorescence channel (excitation 470/22 nm and emission 510/42 nm) whereas Iba1 and BDNF were imaged under red fluorescence channel (excitation 531/40 and emission 593/40). Similar observations were noted in triplicate wells/group in three independent experiments. Scale bar = 100 μm. All images were cropped at the same scale using Adobe Photoshop CC 19.1.9 for clarity and conciseness of the presentation. Full-length images of a representative set (B) is presented in S1 Fig.