Baby lotion

WT, TNFR1KO, TNFR2KO, and TNFR1; TNFR2-double-null mice (DKO) are in the C57BL/6 background. 8–10-week-old mice with equal number of males and females (n=6) received a daily topical dose of 80 mg (4 mg of the active compound) of commercially available imiquimod cream (Aldara, 3M Health Care Limited, UK).
The cream was applied on the shaved (using a Wahl Pocket Trimmer) dorsal skin (~4 cm² area) for 4 consecutive days as reported earlier (van der Fits et al., 2009 (link)). Mice were euthanized and skin and blood samples were collected. Control mice were treated with a baby lotion (Johnson & Johnson), which contains similar inactive ingredients as Aldara cream. The harvested skin specimens from each mouse are divided into 4 aliquots for paraffin fixation, frozen sections, RNA and protein extraction.

Sterile saline or ET-1 (Enzo Life Sciences, Farmingdale, NY, USA) dissolved in deionized water was administered (5.25 pmol) in 10 μL (1.31 ng/μL) subcutaneously using an insulin syringe into the left plantar hindpaw on postnatal day 7 (P7). Animals were videotaped, and the number of spontaneous paw flinches was counted. On postnatal day 11 (P11), capsaicin cream (0.1% Capzaisin-HP, Chattem, Inc, Chattanooga, TN, USA) or control lotion (Johnson & Johnson Baby Lotion) was applied topically to the dorsum of the right hindpaw. There were four treatment groups for both sexes (n = 5–6 for each group and sex): saline (P7) + control (P11), saline (P7) + capsaicin (P11), ET-1 (P7) + control (P11), and ET-1 (P7) + capsaicin (P11). Before application of capsaicin, the baseline paw withdrawal threshold was measured in the plantar right hindpaw using sequential von Frey filaments ranging from 0.04 g to 1.4 g. Each filament was applied a total of five times and the first filament that elicited a sustained response was considered the paw withdrawal threshold. This method was used at 20, 40, 60, and 120 min time points following capsaicin application.