Biotin blocking buffer

After three PBS washes, the sections were permeabilised and blocked by incubation with 10% goat serum, 0.3% Triton-X in PBS for 120 min at RT, followed by biotin blocking buffer (Vector Laboratories). The sections were then incubated in a humidity chamber overnight at 4°C with anti-ERα antibody (polyclonal, made in rabbit, Abcam ab75635), or normal rabbit serum was diluted 1:100 in blocking buffer. Next, the sections were washed in 0.3% Tween 20 PBS and were incubated at room temperature for 60 min with biotinylated goat anti-rabbit IgG (Vector Laboratories), diluted 1:200 in 1% normal goat serum. Detection was performed with the Vectastain ABC kit (Vector Laboratories) according to the manufacturer’s instructions.

after three PBS washes, the sections were permeabilised in 0.2% Triton X-100 in PBS and blocked by incubation with 10% goat serum and 0.3% Tween 20 in PBS for 30 min at 37°C, followed by biotin blocking buffer (Vector Laboratories). The sections were incubated in a humidity chamber overnight at 4°C with rabbit anti-luciferase antibody or normal rabbit serum diluted 1:6000 in blocking buffer. The anti-luciferase antibody was provided by W. Just (Soto, et al. 1993). Next, the sections were washed in 0.3% Tween 20 PBS and were incubated at room temperature for 60 min with biotinylated goat anti-rabbit IgG (Vector Laboratories) and diluted 1:200 in 1% normal goat serum 0.3% Tween 20. Detection was performed with the Vectastain ABC kit (Vector Laboratories) according to the manufacturer’s instructions. The sections were counterstained with haematoxylin/eosin.