Cy3 conjugated anti mouse igg

The rabbit polyclonal anti-SPARC antibody (15274-1-AP) was purchased from Proteintech. The mouse monoclonal anti-human SPARC (clone AON-5031, sc-73472), the rabbit polyclonal anti-human cath-D antibody (H-75, sc-10725), and the mouse monoclonal anti-human cath-D (clone C-5, sc-377124) antibodies were purchased from Santa Cruz Biotechnology. The mouse monoclonal anti-human cath-D antibody (clone 49, #610801) was purchased from BD Transduction Laboratories^TM, and the goat polyclonal anti-mouse cath-D (AF1029) from R&D Systems. The anti-human cath-D antibodies M1G8 and D7E3 were previously described 19 (link). The mouse monoclonal anti-tubulin antibody (clone 236-10501, #A11126) was from Thermo Fisher Scientific, the mouse monoclonal anti-Myc (clone 9B11) from Ozyme, and the rabbit polyclonal anti-β actin antibody (#A2066) from Sigma-Aldrich. The horse anti-mouse immunoglobulin G (IgG)-horseradish peroxidase (#7076), and goat anti-rabbit IgG-HRP (#7074S) secondary antibodies were from Cell Signaling Technology. The donkey anti-goat HRP conjugated antibody (FT-1I7890) was from Interchim. The Alexa Fluor 488-conjugated anti-rabbit IgG (#Ab150077) was purchased from Abcam, and the Cy3-conjugated anti-mouse IgG (#SA00009.1) from Proteintech. Hoechst 33342 (#FP-BB1340) was from Interchim FluoProbes.

Rats were anesthetized and perfused through the ascending aorta with NS, followed by 300 mL of 4% paraformaldehyde in 0.1 M phosphate buffer. Lumbar enlargement segments were then harvested, postfixed at 4 °C overnight, and dehydrated in 30% sucrose. Afterwards, 30-μm free-floating transverse sections were cut at −20 °C in a freezing microtome. Sections were collected in phosphate-buffered saline and double-labeled to investigate the co-expression of NR2B and neuronal, glial, or microglial markers. Specifically, spinal sections were incubated for 48 h at 4 °C in a mixture of mouse anti-NR2B (1:200; Abcam) and rabbit anti-PSD-95 (1:2000; Abcam), rabbit anti-NeuN (1:500; Abcam), rabbit anti-GFAP (1:500; Proteintech, IL), or rabbit anti-Iba-1 (1:1000; Abcam) antibodies. The sections were then incubated with a mixture of Cy3-conjugated anti-mouse IgG (1:200; Proteintech) and fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:200; Proteintech), for 2 h at room temperature. Fluorescent signal was viewed with a fluorescence microscope (OLYMPUS, x-Cite 120, Japan) with the appropriate filters.