Alexa fluor igg h l secondary antibodies

Mice were anesthetized and then transcardially perfused with 4% paraformaldehyde in phosphate buffered saline (PBS). Optic nerves were dissected and postfixed in 4% paraformaldehyde in PBS for 2 h, and cryoprotected in 30% sucrose in PBS overnight. About 8 mm segments of the optic nerve centered at the injury site were embedded in OCT compound (Tissue-Tek) and longitudinal sections (10 μm) were cut using a cryostat. Sections were immunostained by incubating in primary antibodies in 5% Normal Goat Serum in PBS with 0.3% Triton-X overnight at 4 °C. Primary antibodies used were: RFP (Rockland 600–401-379S, 1:1000), GFAP (Invitrogen 130,300, 1:1000 or Dako Z033429, 1:500), APC/CC1 (Millipore OP80 Ab-7, 1:500), NG2 (Millipore AB5320, 1:200), GFP (Abcam ab13970, 1:2000) and Iba1 (Wako 019–19,741, 1:500). Following primary antibody incubation, sections were washed and incubated in species-appropriate Alexa Fluor IgG (H + L) secondary antibodies (Invitrogen, 1:500) at room temperature for 1 h. For CC1, goat-anti-mouse IgGγ2b secondary antibody was used (Invitrogen A-21141, 1:500). Slides were mounted using Vectashield with DAPI (Vector Laboratories H-1200). Images were obtained using a Nikon Eclipse Ti fluorescent microscope or an Olympus FluoView 1000 confocal microscope.

Mice were anesthetized and then transcardially perfused with 4% paraformaldehyde in phosphate buffered saline (PBS). Spinal cords were dissected and postfixed in 4% paraformaldehyde in PBS for 2 h, and cryoprotected in 30% sucrose in PBS overnight. For spinal cord injury tissue, 8 mm segments of the spinal cord centered at the injury site were embedded in OCT compound (Tissue-Tek) and serial sagittal sections (16 μm) were cut using a cryostat and thaw mounted onto slides. For EAE tissue, 3 mm segments of cervical, thoracic, and lumbar spinal cord were embedded in OCT compound and serial cross sections (16 μm) were cut using a cryostat and thaw mounted onto slides. Sections were immunostained with primary antibodies in 5% Normal Goat Serum in PBS with 0.3% Triton-X overnight at 4 °C. Primary antibodies used were: RFP (Rockland 600–401-379S, 1:1000), GFAP (Invitrogen 130300, 1:1000; Abcam ab4674, 1:1000), APC/CC1 (Millipore 0P80 Ab-7, 1:500), GFP (Abcam ab13970, 1:2000), PDGFRβ (Abcam ab32570, 1:500), and p75 (Neuromics GT15057, 1:500). Following primary antibody incubation, sections were washed and incubated in species-appropriate Alexa Fluor IgG (H + L) secondary antibodies (Invitrogen, 1:500) at room temperature for 1 h. Slides were mounted using Vectashield with DAPI (Vector Laboratories H-1200).

For Cre-dependent anterograde labeling of Pax7 expressing neurons in the superficial SC (sSC), approximately 100 nL of AAV2–CAG–FLEX–GFP (University of North Carolina Vector Core) was injected into the sSC of Pax7-Cre mice (10 weeks old). Injection coordinates were as follows: posterior from bregma, lateral from midline, and depth in mm, 4.16, 0.2, 0.5, and 1.0–1.2, respectively. Three to 4 weeks after AAV injection, mice were anesthetized and then transcardially perfused with 4% paraformaldehyde in phosphate buffered saline (PBS). Brains were dissected and postfixed in 4% paraformaldehyde in PBS for 16 h, and cryoprotected in 30% sucrose in PBS for 2–3 days. Brains were embedded in OCT compound (Tissue-Tek) and coronal sections (20 μm) were cut using a cryostat. Sections were immunostained by incubating in primary antibodies in 5% Normal Goat Serum in PBS with 0.3% Triton X-overnight at 4°C. Primary antibodies used were: RFP (Rockland 600–401-379S, 1:1000) and GFP (Abcam ab13970, 1:2000). Following primary antibody incubation, sections were washed and incubated in species-appropriate Alexa Fluor IgG (H + L) secondary antibodies (Invitrogen, 1:500) at room temperature for 1 h. Slides were mounted using Vectashield with DAPI (Vector Laboratories H-1200). Images were obtained using a Nikon Eclipse Ti fluorescent microscope or an Olympus FluoView 1000 confocal microscope.