Alexa fluor 488 labeled donkey anti rabbit secondary antibody

For immunofluorescence staining of p-c-jun, cells were cultured in the EpiLife medium with 0.1% DMSO, 5 µg/ml LPS, or 5 µg/ml LPS plus 50 µg/ml RGO for 6 hours. Fixation and blocking procedures were conducted as shown earlier. Cells were incubated with rabbit polyclonal p-c-jun antibody (1:100 dilution; Cell Signaling) at 4℃ overnight, washed thrice with PBS, and incubated with Alexa Fluor 488-labeled donkey anti-rabbit secondary antibody (Molecular Probes, Eugene, OR, USA). Cells were then washed with PBS and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 minutes.

Sebocytes and ORS cells in an 8-well chamber slide (Nunc Lab-Tek, Roskilde, Denmark) were fixed in 4% paraformaldehyde for 10 minutes, blocked with 5% normal donkey serum for 1 hour, and then incubated with antibody dilutions of CK1-3 (1:100; Chemicon), CK8 (1:100; Chemicon), CK15 (1:100; Chemicon), CK17 (1:100; Abcam, Cambridge, UK), and CK19 (1:100; Chemicon) at 4℃ overnight. After washing with PBS, the cells were incubated using Alexa Fluor 488-labeled donkey anti-rabbit or mouse secondary antibody (Molecular Probes, Eugene, OR, USA) for 1 hour. Finally, the cells were counterstained using 4,6-diamidino-2-phenylindole (DAPI) for 10 minutes.
For immunofluorescence staining of p-c-jun, cells treated with 5 µg/ml LPS or 5 µg/ml LPS and 50 µg/ml BG for 6 hours were fixed and blocked as above, maintained with rabbit polyclonal p-c-jun antibody dilution (1:100; Cell Signaling) at 4℃ overnight, and incubated using Alexa Fluor 488-labeled donkey anti-rabbit secondary antibody (Molecular Probes). Finally, the cells were counterstained using DAPI for 10 minutes.

The excised mouse skin tissues were embedded in a frozen section compound, OCT compound (Tissue‐Tek; Miles, IL, USA) in a freezer at −80°C. Tissues were cut to a thickness of 8 μm using a cryostat (Leica CM3050 S; Leica, Heidelberg, Germany) and fixed at 4% paraformaldehyde (Sigma, Louis, MI, USA) for 10 min. After blocking in 5% normal donkey serum (Abcam, Cambridge, UK) for 1 h, the primary antibody was incubated overnight at 4°C (loricrin, 1:100 dilution; filaggrin 1:100 dilution; or involucrin, 1:100 dilution; all from Abcam). After washing three times with PBS, Alexa Fluor 488‐labeled donkey anti‐rabbit secondary antibody was used to observe the expression (Molecular Probes, Eugene, OR, USA) and counterstained with 4’6‐diamidino‐2‐phenylindole (DAPI) for 10 min.