Ab44520

Briefly, 5 µm sagittal sections of fixed human lung were deparifinized and rehydrated as discussed earlier. After epitope retrieval, oestrogen receptor (ER) alpha (ERα) (Santa Cruz, sc-7207; 1 µg/mL), ERβ (Abcam, ab-3577; 5 µg/mL), GPER (Abcam, ab-39742; 5 µg/mL), and SERT (Abcam, ab-44520: 1:200) were incubated overnight at 4°C. Lung sections were counterstained with haematoxylin. Distribution was assessed by staining consecutive sections with alpha smooth muscle actin (for medial cells) and von-Williebrand factor (for endothelial cells).

Brain tissues were fixed in paraformaldehyde, embedded, and cut into sections (5 μm thickness). Then tissue sections were dewaxed in xylene and rehydrated in graded alcohols. After that, sections were incubated with 0.3% hydrogen peroxide to inactivate endogenous peroxidase activity. Antigen retrieval was achieved by incubating with 0.1 M sodium citrate (pH 6.0). After blocking with goat serum (Cat# ZLI-9022; Zhong Shan-Golden Bridge Biological Technology CO., Ltd., Beijing, China) for 30 min, sections were incubated with primary antibodies against TPH (1 : 300; ab46200; Abcam), ERβ (1 : 200; ab3577; Abcam), and SERT (1 : 1000; ab44520; Abcam) at 4°C overnight. After washing with PBS, secondary antibodies of FITC tagged goat anti-rabbit IgG (1 : 100; ZF-0311, ZSGB-bio, Beijing) were added and incubated for 30 minutes in the dark. After rinsing with PBS for 3 times, the sections were analyzed on a laser scanning confocal microscope (LSM510, Zeiss, Jena, Germany). Three different visual fields were randomly selected for each target brain area of each slice for photographing. The fluorescence intensity was quantified using Image-Pro Plus 6.0 (Media Cybenetics, Silver Spring, USA).