Cytm 3 conjugated affinipure donkey anti goat igg

Seventy-two hours after H129-H8 injection, mice were anesthetized with pentobarbital sodium (100 mg/kg, i.p.), and perfused transcardially with 30 mL sodium phosphate buffer (PBS) and followed by ice-cold 4% paraformaldehyde (PFA) dissolved in 0.1 M PBS. The brain tissues were carefully removed and post-fixed in PBS containing 4% PFA at 4°C overnight, and then equilibrated in PBS containing 30% sucrose at 4°C for 3 days. The 40 μm thick coronal sections were obtained using the cryostat microtome (Thermo, CRYOSTAR NX50, Cheshire, United Kingdom). Sections containing MePD/MePV were incubated with goat polyclonal antibody against NPSR (1:1,000, sc-162893, Santa Cruz Biotechnology, Santa Cruz, CA, United States) diluted in PBS containing 1% bovine serum for 48 h at 4°C on an agitator after incubation in 10% bovine serum in PBS. After several rinses in PBS, sections were incubated with CyTM 3-conjugated affinipure donkey anti-goat IgG (1:1,000, 705-165-147, Jackson ImmunoResearch Laboratories, Inc., PA, United States) for 4 h at room temperature and then with DAPI (1:2000, D8417, Sigma-Aldrch, Saint Louis, MO, United States) for 10 min. Finally, sections were on slides, covered with a coverslip, mounted with 70% glycerol (in PBS) and sealed with nail polish. Images were captured with the virtual microscopy slide-scanning system (VS120, Olympus, Tokyo, Japan).

These sections were incubated with mixture solution including rabbit polyclonal antibody against c-Fos (1:1,000, sc-253, Santa Cruz Biotechnology, Santa Cruz, CA, United States) and goat polyclonal antibody against NPSR (1:1,000, sc-162893, Santa Cruz Biotechnology, Santa Cruz, CA, United States) diluted in PBS containing 1% bovine serum for 48 h at 4°C on an agitator after incubation in 10% bovine serum in PBS. The specificity of the anti-NPSR antibody has been demonstrated in previous studies (Laitinen et al., 2004 (link); Vendelin et al., 2005 (link); Shao et al., 2013 (link), 2016 (link); Xie et al., 2018 (link); Wang et al., 2020 (link)). After several rinses in PBS, sections were incubated with Alexa Fluor^® 488-conjugated affinipure donkey anti-rabbit IgG (1:1,000, 711-545-152, Jackson ImmunoResearch Laboratories, Inc., PA, United States) and CyTM 3-conjugated affinipure donkey anti-goat IgG (1:1,000, 705-165-147, Jackson ImmunoResearch Laboratories, Inc., PA, United States) for 4 h at room temperature. Finally, sections were mounted on slides, covered with a coverslip, using 90% glycerol in 0.1 M PB, and observed under a fluorescence microscope (BX53, Olympus, Tokyo, Japan) and photographed under Zeiss LSM 710 laser confocal microscope.