All primary antibodies were diluted in blocking buffer and incubated at 4°C for 18 h.
Anti-GPER (MBL, Woburn, MA) was diluted 1∶250 in blocking buffer and incubated overnight at 4°C.
Primary antibodies were diluted in the blocking buffer and incubated with cells for 45 min at RT.
Primary antibody for actin was diluted 1∶10000, while α-, γ-tubulin, and LDLR antibodies were diluted 1∶1000 in blocking buffer and incubated overnight at 4°C.
The blocking buffer then was replaced with HRP-6B6C.1 antibody diluted in blocking buffer and incubated for 1 h at room temperature.
Then, wells were treated with blocking buffer, washed and samples were serially diluted in blocking buffer and incubated 2 hours at 37°C.
Secondary HRP- or AP-conjugated antibodies were diluted 1/5000 in the respective blocking buffer and incubated with blots for 30 60 minutes.
Alexa fluor 647 labeled streptavidin was diluted 1 500 in blocking buffer and incubated with the cells for 30 60 mins at room temp.
For this purpose sections were washed in TBS, re-blocked for 20 min in blocking buffer and incubated overnight at 4°C with 1∶1500 diluted sheep anti-TH antibody (Pel-Freeze, AR).
Secondary antibodies were diluted in blocking buffer and incubated for 1 h at RT. Horseradish peroxidase-conjugated goat anti-rabbit antibody (1∶5000 dilution) was used to detect labeled product.
Preparations were then washed twice 15 min followed by a 30 min rinse in blocking buffer and incubated with the secondary antibodies for 80 min at room temperature in a humidified atmosphere.
