Trpa1

The immunohistochemical staining protocol was carried out following the protocol of our previous study [26 (link)]. Primary antibodies: TRPV1 (rabbit polyclonal antibody, 1 : 100, Alomone Labs, Israel), TRPA1 (rabbit polyclonal antibody, 1 : 100, Alomone Labs, Israel), and TRPM8 (rabbit polyclonal antibody, 1 : 100, Alomone Labs, Israel); and secondary antibody (goat-anti-rabbit 1 : 100, Boster, China) was used and stained with DAB (Dako, Denmark). The percentage of immunopositive cells was analyzed by counting the number of immunopositive neurons and multiplying by 100/number of total number of neurons. A total of 2 DRGs were measured in each animal, and the number of immunoreactive neuronal profiles was counted in a blinded fashion. The percentage of immunopositive cells was used for statistic index.

Slides were deparaffinized, rehydrated, and heated in citrate buffer pH 6 for antigenic retrieval. After blocking for endogenous peroxidase with 3% hydrogen peroxide, the following antibodies were incubated: TRPA1 (#ACC-037 1:500, 1 h, Alomone Labs, Jerusalem, Israel), TRPV2 (SAB1101376 Sigma, 1:1000, 1 h), TRPC3 (#ACC-016 Alomone, 1:500, 1 h), TRPC6 (#ACC-017 Alomone, 1:300, 1 h). Immunohistochemistry was performed using the streptavidin-biotin-peroxidase method with diaminobenzidine as the chromogen (Kit LSAB, Dakocytomotion, Glostrup, Denmark). Slides were finally counterstained with haematoxylin. Negative controls were obtained after omission of the primary antibody or incubation with an irrelevant antibody. For cryo-sections, retinas were fixed overnight with 4% PFA, rinsed in PBS and then incubated with 30% sucrose before OCT embedding. Retina sections were blocked and permeabilized in PBS 0.25% triton, 5% FBS, 1% BSA for 2 h and incubated overnight at 4 °C with the rabbit anti-TRPA1 antibody (#ACC-037 Alomone, 1:100) and the rat anti-CD31 (Becton Dickinson, 1/100). After washes, sections were incubated with secondary antibody donkey anti-rabbit A488 and donkey anti-Rat A594 for 2 h at room temperature. Sections were then washed and stained with DAPI before mounting in Mowiol. Sections were imaged with a Carl Zeiss AxioImager Z1-Apotome.

Mice were sacrificed by cervical dislocation under ether anesthesia 15 days after the WTD and ibuprofen administration. Plantar surfaces of injured hind paw skin were dissected and stored at −80°C for western blot analysis. The expression of TRPV1, TRPA1, and TRPM8 in hind paw skins tissues was determined. The western blot protocol and semiquantitative analysis were carried out following the protocol of our previous study [26 (link)]. The following primary antibodies were used: TRPV1 (rabbit polyclonal antibody, dilution 1 : 100, Alomone Labs, Israel), TRPA1 (rabbit polyclonal antibody, dilution 1 : 100, Alomone Labs, Israel), TRPM8 (rabbit polyclonal antibody, dilution 1 : 100, Alomone Labs, Israel), and GAPDH (internal control, rabbit polyclonal antibody, dilution 1 : 1000, Beyotime, China). A goat-anti-rabbit antibody conjugated to horseradish peroxidase (1 : 5000, Thermo Fisher Scientific, USA) was used as the second antibody. TRPV1, TRPA1, and TRPM8 protein levels were normalized against GAPDH. All experiments were done for four times.