Immunofluorescence staining was performed on hDPCs and 5-μm frozen sections of hair follicles. The hDPCs were plated in 8 chamber slides (LAB-TEK, Rochester, NY, USA) at a density of 1.6 × 103 cells per well and cultured in serum-free DMEM with 200 ng/ml HMGB1 or with blocking antibodies.
Hair follicles and hDPCs were fixed in 4% paraformaldehyde for 15 min, and then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) and blocked with 10% goat serum. The following primary antibodies were used: anti-Ki-67 (DAKO, Carpinteria, CA, USA), mouse anti-COX-1 (1:100; Abcam, Cambridge, MA, USA), FITC-conjugated anti-COX-2 (1:100; Abcam), rabbit anti-mPGES-1 (1:100; Cayman Chemicals) and rabbit anti-mPGES-2 (1:100; Cayman Chemicals). After washing with PBS, samples were incubated with the appropriate secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 635 goat anti-mouse IgG (Life Technologies, Eugene, OR, USA) and Alexa Fluor 635 goat anti-rabbit (Abcam). Slides were washed in PBS and counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Life Technologies). Immunofluorescence images were acquired using Zeiss LSM 700 confocal laser scanning microscopy software (Carl Zeiss, Oberkochen, Germany). For quantitative analyses, Ki-67-positive cells were counted and normalized to the number of DAPI-stained cells using ZEN 2009 software (Carl Zeiss).
Hair follicles and hDPCs were fixed in 4% paraformaldehyde for 15 min, and then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) and blocked with 10% goat serum. The following primary antibodies were used: anti-Ki-67 (DAKO, Carpinteria, CA, USA), mouse anti-COX-1 (1:100; Abcam, Cambridge, MA, USA), FITC-conjugated anti-COX-2 (1:100; Abcam), rabbit anti-mPGES-1 (1:100; Cayman Chemicals) and rabbit anti-mPGES-2 (1:100; Cayman Chemicals). After washing with PBS, samples were incubated with the appropriate secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 635 goat anti-mouse IgG (Life Technologies, Eugene, OR, USA) and Alexa Fluor 635 goat anti-rabbit (Abcam). Slides were washed in PBS and counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Life Technologies). Immunofluorescence images were acquired using Zeiss LSM 700 confocal laser scanning microscopy software (Carl Zeiss, Oberkochen, Germany). For quantitative analyses, Ki-67-positive cells were counted and normalized to the number of DAPI-stained cells using ZEN 2009 software (Carl Zeiss).