Alexa fluor 488 conjugated donkey anti goat antibody

Immunostaining protocol was adapted from a published protocol [19 (link)]. Mouse eyes were enucleated and fixed in 4% PFA at room temperature for 15 minutes, then transferred to PBS for 10 minutes. After whole eye fixation, retinas were dissected and fixed with cold methanol (-20°C) for 20 minutes, followed by blocking in Perm/Block solution (PBS + 0.3% Triton + 0.5% BSA + 5% goat serum). Goat anti-mouse PECAM-1 (1:50) (R&D Systems, Fischer Scientific, #AF3628) combined with Alexa Fluor 594-conjugated isolectin GS B4 (1:200) (Invitrogen, #I21413) in Perm/Block solution was used for primary antibody incubation, followed by Alexa Fluor 488-conjugated donkey anti-goat antibody (1:1000) (Invitrogen, #A32814) for secondary antibody incubation. After extensive washing, retinas were mounted using ProLong Glass Antifade (Invitrogen) onto a coverslip and allowed to set overnight. Immunofluorescence was captured via Olympus Provis AX-70 and Leica TCS SP8 Confocal microscopes. The fluorescence intensity was quantified using ImageJ software.

Glass coverslips were placed in a 24-well plate and 2 × 10⁴ cells/500 μL of BeWo WT, BeWo KO, and BeWo KO-HtrA4 rescue cells were seeded. The cells were exposed to 50 μM forskolin or equal amounts of DMSO for 48 h. After treatment, the cells were fixed using 4% paraformaldehyde for 10 min at room temperature (RT) and washed twice with phosphate-buffered saline (PBS) for 2 min each time. The cells were then permeabilized with 0.2% triton X-100 for 1 min at RT, blocked with PBS containing 1% BSA for 1 h at RT, and incubated with E-cadherin and HCG antibodies (Table S3) at 4 °C overnight. After washing with PBS, the cells were incubated with Alexa-fluor-488-conjugated donkey anti-goat antibody (Invitrogen, Carlsbad, CA, USA) and Alexa-fluor-568-conjugated goat anti-rabbit antibody (Invitrogen, Carlsbad, CA, USA) at RT for 1 h. The cell-cell fusion was examined under a confocal microscopy (Zeiss LSM880, Carl Zeiss Microscopy GmbH, Munich, Germany).

Animals were anesthetized and transcardially perfused with saline followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Brains were removed, postfixed in 4% paraformaldehyde in PBS for 24 h at 4°C and sectioned on a vibrating microtome (VT1000S, Leica) into 50 μm thick coronal sections (stereotaxic coordinates: dorsoventral −8 mm, lateromedial ±0.4 mm, and anteroposterior −1.88 mm from bregma). Free-floating sections were washed in PBS and incubated in 5% normal donkey serum (NDS, Sigma-Aldrich) and 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 1 h. They were then incubated (48 h, 4°C) in PBS containing goat anti-CRH antibodies (1:100; Santa Cruz Biotechnology: sc-1759). After several washes the sections were incubated in PBS with Alexa Fluor 488-conjugated donkey anti-goat antibodies (1:200, 2 h, Thermo Fisher Scientific). Finally, the sections were mounted onto gelatin-coated slides and coverslipped using PBS-buffered glycerol. The sections were analyzed and photographed using a Leica DM 6000 B microscope equipped with a digital camera (MBF CX9000, Williston, VT USA; Figure 1B).

After 1–2 days in culture, except where noted, wt and GCaMP6f-expressing murine ACC were rinsed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS for 10 min. Cultures were blocked in PBS containing 0.1% Triton X-100 (PBST) and 10% fetal bovine serum (FBS) and incubated overnight at 4°C with antibodies against TH (rabbit, PA5-85167, ThermoFisher, Waltham, MA, USA), phenylethanolamine N-methyltransferase (PNMT) (rabbit, AB110, MilliporeSigma, Burlington, MA, USA), or S100β (rabbit, GA50461-2, Agilent, Santa Clara, CA, USA). For GCaMP6f-expressing cells, the overnight incubation included a green fluorescent protein (GFP) antibody (goat, 600-101-215, Rockland Immunochemicals, Pottstown, PA, USA). Primary antibodies were diluted 1:1000. Cells were then rinsed several times with PBS and incubated for one hour at room temperature in the dark with AlexaFluor 594-conjugated donkey-anti-rabbit and/or AlexaFluor 488-conjugated donkey anti-goat antibodies (ThermoFisher). Secondary antibodies were diluted 1:500 in 10% FBS in PBST that also contained 1 μg/ml bisbenzimide (Hoechst 33342; ThermoFisher) to label nuclei. Cells were rinsed with PBS and brightfield and fluorescence images were acquired using a Nikon TE 2000 epifluorescence microscope equipped with an iXonEM + DU-897 EMCCD camera (Andor).