Since antibodies to the oestrogen receptor were raised in the same species, Anti‐ERα (Saint John's Laboratory, UK) and Anti‐GPER1 (Saint John's Laboratory, UK) were conjugated to fluorescein isothiocyanate (FITC) (Thermofisher, UK), as per the manufacturers’ protocols (Thermofisher UK). Briefly, Anti‐ERα and Anti‐GPER1 were initially concentrated using Amicon Ultra centrifugal filter units (Ultra 4 MWCO 30KDA) (Merck Millipore, UK) to generate stock solutions of concentrated Anti‐ERα and Anti‐GPER1 at 2 mg/ml in 0.1 M sodium bicarbonate. FITC stock solution was prepared at 1 mg/ml in anhydrous dimethyl sulphoxide (DMSO). Then, 50 μl of FITC solution was slowly add to the 2 mg/ml of antibody solution and incubated at 4°C for 1 h with continuous stirring. Free fluorophores were separated from conjugated antibody using PD‐10 column, Sephadex G‐25 M (BioRad, UK) and conjugated ERα/ FITC and GPER/FITC was eluted with PBS. In order to determine the labelling efficiency and concentration, absorbance values were measured at A280 and A495. For effective labelling, the degree of labelling should fall within 2–6 mol of FITC per 1 mol of antibody; typical concentrations were in the range of 0.2 mg/ml. The newly conjugated antibody was stored at −20°C until use.
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