Fitc conjugated goat anti rabbit secondary antibody

For cells suspension culture, cells were plated in 100 mm non-adhesion culture dish (JET BIOFIL) over 5 days, regularly change the medium and verified by ALDH1 expression through flow cytometry detection as above. ZNF32 and GPER expression detection in suspension or normal cultured cells were examined by flow cytometry (BD FACSAria, Franklin Lakes, NJ, USA), according to the manufacturer’s recommended protocol. ZNF32 antibody was produced and purified as previously described^{27 (link)}. GPER antibody was purchased from Santa Cruz Biotechnology (Santa, sc-48525-R #B0216 USA). Rhodamine (TRITC) -conjugated AffiniPure Donkey Anti-Mouse (H + L) antibody was purchased from BBI Life Sciences (#D1100883–0100, China). FITC Conjugated Goat Anti-Rabbit Secondary Antibody was purchased from BOSTER (#BA1105, China). Individual fluorescent populations were determined using acquisition and analysis software (FlowJo 7.6).
A detailed description of the mammosphere assay protocol for the quantification of breast stem cell activity is described in a recent publication^{3 (link),28 (link)}. Briefly, cells were plated in suspension culture at 500 cells/cm². Mammosphere forming was calculated by dividing the number of mammospheres (colonies > 60μm in diameter) formed by the number of cells plated and expressed as a number.

RAW 264.7 cells were fixed using pre-cold acetone, and then rinsed with PBS three times. The cells were permeabilized in 0.1% Triton X-100 and incubated with 1% BSA/PBS. Then, cells were immunostained by incubating with antibody against MMP-2, MMP-9 or TNFRSF1B (diluted 1:500, Santa Cruz Biotechnology, Dallas, Texas, USA) overnight at 4 °C. After being washed with PBS, cells were incubated with the FITC-conjugated goat anti-rabbit secondary antibody (diluted 1:60, Boster Biotechnology, Wuhan, Hubei, China). Nuclei were stained with DAPI (Biotime Biotech, Haimen, Jiangsu, China). Images were taken on a Zeiss inverted microscope (Carl Zeiss, Hallbergmoos, Germany).

The BECs were fixed by 95% ethanol and 0.1% Triton-X100. The samples were then blocked with normal goat serum for 20 min and incubated overnight with SP (Bioss, bs-0065R, Beijing, China), VIP (Bioss, bs-0077R, Beijing, China), or CGRP (Bioss, bs-0791R, Beijing, China) rabbit primary antibody at 4°C. Then, the samples were incubated with a FITC-conjugated goat anti-rabbit secondary antibody (Boster, BA1105, Wuhan, China) at room temperature for 1 h. After counterstained with DAPI for 10 min, the samples were observed with a laser scanning confocal microscopy. Images were acquired with ×40 objectives using a digital camera. The integral optical density values (IOD) of the VIP, SP, and CGRP protein were measured by Image-Pro Plus 6.0.