Alexa 488 conjugated donkey anti goat igg

After cryosection, 2 μm thick brain sections were mounted onto glass slides. Sections were washed with PBS (pH 7.4), followed by antigen retrieval using sodium citrate buffer, pH 6, at 95°C for 25 min in a water bath. Nonspecific staining in sections was blocked using 10% donkey serum in 0.1% Tween 20-PBS for 1 h at room temperature. Primary antibodies were added overnight at a dilution of 1 : 350 for goat Iba-1 (Abcam), 1 : 1,000 for rabbit MMP-2 (Abcam) at 4°C. Alexa 488-conjugated donkey anti-goat IgG (1 : 200, Jackson Lab, Suffolk, United Kingdom) or Cy3-conjugated donkey anti-rabbit IgG (1 : 200, Jackson Lab) was subsequently applied. The nuclei were counterstained with DAPI (Sigma-Aldrich). Images were taken using a microscope (Axio Imager Z1, Carl Zeiss, China). This part was similar with the methods of Hu et al. [32 (link)].

BM cells were fixed with 4% paraformaldehyde for 20 min at room temperature and the cells were rinsed and suspended in the culture medium. 1×10⁶ cells were pelleted in microfuge and resuspended in 0.2 mL of deionized H₂O. 0.1 mL of the cell suspension was added to a chamber (48 mm²) in 3-chamber cytofuge cassette (StatSpin Cytofuge 2, Nihon Rufuto, Tokyo, Japan) on MAS-coated slide glass (Matsunami Glass, Osaka, Japan), and settled onto glass surface by centrifugation at 1000 rpm for 4 min. The cells were briefly dried on a hot plate at 30 ˚C. The cells were then immersed in PBS, incubated in a blocking buffer (5% donkey serum, 0.1%TX100, PBS) for 45 min at room temperature, and reacted with goat anti mouse EpoR antibody (AF1390, R&D Systems, 1/200 in the blocking buffer) at 4˚C overnight. The primary antibody was detected by Alexa488-conjugated donkey anti-goat IgG (Jackson ImmunoResearch, West Grove, PA). After mounted in VectaShield with DAPI (Vector Laboratories), images were taken by LSM800 confocal microscope (ZEISS, Oberkochen, Germany). Images were analysed by using Image J (https://imagej.nih.gov/ij/index.html).