Perfusion and section preparation

Perfusion and section preparation

One hour after the blue illumination (473 nm laser Shanghai Laser & Optics Century Co. Ltd, China, 30Hz, 10 ms pulse width, 40 mW/mm² ) parallelly with the repeated mechanical stimulation (0.16 g von Frey filament every 30 s for 20 min), mice were sacrificed and perfused transcardially with 20 ml PBS and following 20 ml 10% formalin fixative solution (Merck). Brains were dissected and post-fixed at 4 °C overnight in 10% formalin, then were cut coronally with the vibratome stage (Leica VT1000S, Germany) the next day. Coronal sections (50 μm) were collected and stored in 0.5% formalin at 4 °C for further immunohistochemistry staining.

Immunohistochemistry and quantification

Floating sections of an experimental cohort were processed parallelly under identical conditions. For each round of staining, a negative control was parallelly applied by omitting the primary antibody. For immunohistochemical staining, sections were assembled in a 24-well plate (3 - 4 sections per well) and incubated with 500 μl antigen retrieval buffer (10 mM of trisodium citrate, pH 6, AppliChem, Darmstadt, Germany) for 20 min at 85 °C. Then sections were transferred to room temperature, incubated once in 50 mM glycine (AppliChem) for 15 min, rinsed for 5 min in PBS and then 10 min in PBST (0.2% Triton X-100 (Carl Roth) in PBS). Before incubating with the primary antibody, 4% horse serum (Abcam) in PBS was employed to block non-specific antigens for 45 min. 

For the Fos protein labelling, the rabbit anti-Fos primary antibody (Abcam, ab190289) was used at 1:1000 dilution and sections were incubated over 20-24 h on a shaker at 4 °C. At the second day, sections were rinsed with blocking solution three times (10 min) and then incubated with the secondary antibody (Donkey anti-rabbit Alexa 488, 1:700) at room temperature for 2 h. After rinsing three times (10 min) with the blocking solution, sections were then incubated with Hoechst 33342 (1:10000 diluted in PBS, Molecular Probes) for 10 min and rinsed in PBST three times for 10 min. Finally, sections were rinsed with 10 mM TRIS/HCl (Carl Roth) for 10 min, mounted on slides with Mowiol (Carl Roth) and kept aside for 2 - 3 days for the Mowiol to dry.