Faramount mounting media

Formalin fixed paraffin-embedded sections were de-paraffinized and rehydrated as follows: 100% Histo-Clear (National Diagnostics) for 5min, 100% ethanol for 1 min, 90% ethanol for 1 min, 70 % ethanol for 1 min and finally in PBS for 1 min. Before immunofluorescent staining, an antigen retrieval step was performed by incubating sections in 10 mM Sodium Citrate Buffer (10 mM Sodium Citrate Buffer, 0.05% Tween 20, pH 6.0) at 95° C for 1 hour. After cooling to room temperature for 30 min, slides were washed with PBS and blocked in blocking buffer (1X PBS, 10% goat serum, 0.5% TX- 100, 1% BSA) for 1 hour at room temperature. Sections were incubated with primary antibodies overnight at +4°C. β-III tubulin antibody (Abcam, cat# 78078) was used at a dilution of 1:250, while anti-TRPV1 antibody (Alomone labs, cat# ACC-030) was used at 1:100 dilution. Slides were washed three times in PBS for 5 min each and incubated in secondary antibodies and Hoescht (1:10000, Invitrogen) at room temperature. Slides were washed three times in PBS for 5 min each and coverslips were mounted by using Faramount Mounting media (Dako). Immunostained sections were analyzed on an Olympus FV1000 confocal microscope equipped with a laser scanning fluorescence and a 12 bit camera. Images were taken using a 60x oil PlanApo objective (with and without zoom feature).