Lsm700

About 24 hrs after fixation (4% paraformaldehyde (PFA) in PBS), fish were washed in PBS + 0.25% Triton (PBT), incubated in 150 mM Tris-HCl at pH 9 for 15 min at 70 °C (antigen retrieval), washed in PBT, permeabilized in 0.05% Trypsin-EDTA for 45 min on ice, washed in PBT, blocked in blocking solution (10% goat serum, 0.3% Triton in Balanced Salt Solution, or 2% BSA in PBS, 0.3% Triton) for at least an hour and then incubated in primary (rabbit anti-pERK, Cell Signaling #4370 and/or mouse anti-ERK (tERK), Cell Signaling #4696, 1:500) and AlexaFluor secondary antibodies (1:500) for up to 3 days at 4 °C diluted in blocking solution. In-between primary and secondary antibodies, fish were washed in PBT and blocked for an hour. If necessary, pigmented embryos were bleached for 5 min after fixation with a 5%KOH/3%H₂O₂ solution.
The protocol was similar for dissected brains, except that the brains were dissected in PBS after 24 h of fixation, and the permeabilization step in Trypsin-EDTA and Tris-HCL antigen retrieval was omitted. Dissected brains were mounted ventral toward the coverslip on slides in 70% glycerol prior to imaging. Confocal images of dissected brains were obtained using either a Zeiss LSM 700 or Olympus FV1000.

Cultured HDFs and KFs were washed twice with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 10 min at room temperature, and permeabilized by incubating for 15 min in 0.01% Tween® 20 in PBS. The samples were blocked with 5% bovine serum albumin and incubated with antibodies to HMGB1 (1:1000; Abcam, Cambridge, UK) overnight at 4 °C. The next day, the cells were washed with PBS and incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, Life Technologies, Grand Island, NY, USA) and Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (Invitrogen) for 2 h at room temperature. The cells were mounted on slides using Vectashield® mounting medium with 4′, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories Inc., Burlingame, CA, USA) and viewed using a confocal microscope system (LSM700, Olympus Corp., Center Valley, PA, USA).

On days 7 and 14, wound tissue specimens harvested from the rat model were used for IHC fluorescence evaluation. To visualize immunofluorescence, sections were incubated with antibodies against Ki-67 (1:300 dilution, GB13030-2; Google Biotechnology) and proliferating cell nuclear antigen (PCNA, 1:200 dilution, GB11010; Google Biotechnology) overnight. After rinsing with PBS, sections were incubated with a fluorescent rabbit secondary antibody for 40 min. After washing four times with PBS, cell nuclei were stained with DAPI, and images were acquired with a confocal laser scanning microscope (Olympus LSM 700).