Immunohistochemical Analysis of Tubulin β-3

Paraffin-embedded, fixed tissue blocks were prepared and 3–5 μm sections were stained with haematoxylin and eosin as described [19 (link),32 (link)]. For tubulin β-3 immunohistochemistry, sections were subjected to heat-induced epitope retrieval by incubation in 10 mM sodium citrate, 0.05% Tween20 for 30 min at 95°C then cooled and rinsed in distilled water. Sections were blocked with 10% sheep serum and 1% BSA in TBS for 30 min then incubated at 4°C overnight with 1 μg ml^-1 rabbit polyclonal anti- tubulin β-3 IgG (Biolegend) and 1% BSA in TBS. Sections were then washed with 0.025% Triton X-100 in TBS and endogenous peroxidase activity was quenched with 3% H₂O₂ for 30 min. Bound primary antibody was labelled with excess volume of HRP polymer anti-rabbit IgG reagent (Vector Labs) with 1% BSA in TBS for 30 min. Slides were then washed as previously and incubated with DAB (Thermo) for 5 min. Sections were counterstained with haematoxylin and mounted with DPX.
Images were acquired using a Leica DFC295 camera attached to a Leica DM3000 microscope. For analysis of inflammation, nuclei were counted automatically using the Leica Application Suite V4.5 software (Leica). DAB intensity was analysed as integrated density in ImageJ.