Pe mouse anti rat cd45ra antibody

Sham-operated and injured rats with the treatment of Ibrutinib or vehicle were euthanized at 1-week post-injury or sham operation. Spleen samples were collected and processed for measuring the population of splenic CD45RA+ B cells using PE mouse anti-rat CD45RA antibody (Cat# 551402, BD Bioscience) and flow cytometry. Immediately after deep anesthesia, the spleen samples were collected. The spleen samples were removed and minced in a 3.5 cm-dish with Hank’s Balanced Salt Solution (HBSS, Invitrogen), then transferred to a 50 mL-tube with HBSS. The splenic cell samples were then passed through 40-μm nylon cell strainer to obtain a single-cell suspension as previously described [17 (link)]. Red blood cells (RBCs) in the resulting splenic cells were lysed using RBC lysis buffer (eBioscience). After washing, the splenic cell samples were resuspended in 5 mL of RPMI 1640 (Invitrogen). Flow cytometry system (Sony SY3200, Cell Sorter, Sony Biotechnology iCyT, San Jose, CA, USA, using the Core Facility) was used to measure the populations of CD45RA+ B cells in the splenic cell samples using antibodies against CD45RA (BD Bioscience) according to the manufacturer’s instructions and our recent study [17 (link)]. The population of CD45RA-positive B cells was automatically calculated as the percentage of specific CD45RA-positive cells.

Ibrutinib was purchased from MedChemExpress LLC (Monmouth Junction, NJ, USA). Anti-IgG and anti-Syndecan-1 (CD138, ab60199) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-phospho-BTK-Y223 antibody (5082), Total BTK (D3H3) antibody (5847), and anti-GFAP (D1F4Q)XP (12389) antibody were purchased from Cell Signal Technology. PE mouse anti-rat CD45RA antibody was purchased from BD Bioscience. Anti-GAPDH antibody and Iba1 antibody (SAB2702364) for Western blot were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Iba1 monoclonal antibody (MA5-27726), phosphor-BTK (Tyr223) polyclonal antibody (PA5-105619), goat-anti mouse Alexa Fluor 488 antibody (A-31620) and Donkey anti-rabbit Alexa Fluor 594 antibody were purchased from Fisher Thermo Scientific. Goat anti-mouse IR Dye 680 antibody, goat anti-rabbit IR Dye 680 antibody, goat anti-mouse IR Dye 800 antibody, and goat anti-rabbit IR Dye 800 antibody were purchased from Li-Cor.