Alexa fluor 488 labeled donkey anti goat

Samples (COCs or FGCs) were fixed in 4 % paraformaldehyde (Solarbio) for 2 h in room temperature (RT, approximate to 25 °C), permeabilized in PBS containing 0.1% Triton X-100 for 10 min at RT, and were then blocked in PBS containing 3% BSA for 2 h. The fixed and permeabilized samples were incubated with goat GPR30 antibody (Abcam, ab118512) and/or rabbit LAMP-1 antibody (Proteintech, IL, USA, 55273-1) at a dilution of 1:200 in PBS containing 3% FBS overnight at 4 °C. The target proteins were visualized by incubating with secondary antibodies of Alexa Fluor 488-labeled donkey anti-goat (Abcam, 150129), Alexa Fluor 594-labeled donkey anti-goat (Abcam, ab150132), or Alexa Fluor 488-labeled donkey anti rabbit (Life Technologies, # A-21206) at a dilution of 1:300 at RT for 2 h in the dark. After incubation with antibodies, the nuclei of samples were stained with DAPI (10 μg/mL) for 5 min at RT. The samples were washed with PBS three times after each step. After staining, the samples were mounted on slides, and the slides were observed under a confocal scanning laser microscope (Nikon Eclipse Ti, Tokyo, Japan). Images shown are representative of at least three independent experiments.

The polyclonal rabbit anti‐Sap2 antibody was kindly provided by Prof. Bernhard Hube (HKI Jena, Germany). Polyclonal goat anti‐human C3 was purchased from CompTech, and polyclonal rabbit anti‐MAC was obtained from Abcam. HRP‐labeled rabbit anti‐goat, goat anti‐rabbit, and Alexa Fluor‐488‐labeled donkey anti‐goat antibodies were purchased from Abcam. NHS was collected from five healthy donors, pooled together, and stored at −80 °C for further use.