Donkey anti mouse igg alexafluor647

The following primary antibodies were used in this study: anti-TOMM20 (Santa Cruz Biotechnology; sc-17764, IF 1:1,000), anti-mNG (Chromotek, 32f6; IF 1:500), anti-GM130 (Cell Signaling Technology, #12480; IF 1:1,000), anti-HNRNPA1 (Santa Cruz Biotechnology, sc-32301; WB 1:500), anti-mNG (Cell Signaling Technology, #53061, WB 1:100), and anti-HSP90 (BD Biosciences, 610419; WB 1:5,000). The following secondary antibodies were used: donkey anti-mouse IgG Alexa Fluor 555 (Invitrogen, A32773; IF 1:500), donkey anti-mouse IgG Alexa Fluor 647 (Invitrogen, A32787; IF 1:500), donkey anti-rabbit IgG Alexa Fluor 647 (Invitrogen, A32795; IF 1:500), anti-mouse IgG HRP (Promega, WB 1:10,000), and anti-rabbit IgG HRP (Promega, WB 1:10,000).

Tissue sections were blocked and permeabilized at room temperature for 1 h in 5% bovine serum albumin (Sigma), 0.05% Triton X-100 (Sigma), and 5% heat-inactivated donkey serum (Millipore) or goat serum (Millipore) for microglia or OPC/OL staining, respectively. Antibodies for microglia stains were 1:1,000 rabbit anti-IBA1 (Wako; Richmond, VA), 1:200 mouse anti-iNOS (Invitrogen; San Diego, CA), 1:50 goat anti-Arg1 (NovusBio; Littleton, CO), 1:500 donkey anti-rabbit IgG Alexafluor 546 (Invitrogen), 1:500 donkey anti-mouse IgG Alexa-Fluor 647 (Invitrogen), and 1:250 donkey anti-goat IgG Alexa-Fluor 488 (Jackson Labs). Antibodies for OPC/OL stains were 1:250 rabbit anti-Olig2 (NovusBio), 1:500 mouse anti-APC, clone CC1 (Millipore), 1:500 rat anti-PDGFRα (Invitrogen), 1:500 goat anti-rabbit IgG Alexa-Fluor 647 (Invitrogen), 1:1,000 goat anti-mouse IgG Alexa-Fluor 546 (Invitrogen), and 1:2,000 goat anti-rat IgG Alexa-Fluor 488 (Abcam; Cambridge, MA). Primary antibodies were incubated overnight at 4 °C and secondary antibodies were incubated for 2 h at room temperature. Nuclei were stained with 1:1,000 DAPI (Thermo Fisher Scientific; Waltham, MA) for 10 min at room temperature. Slides were mounted with Mowiol (Sigma), imaged with a Zeiss LSM 880 confocal microscope, acquired using Zen Black software, and analyzed on Bitplane IMARIS software (Concord, MA).