Mouse anti plp

Spinal cords were fixed in 4% paraformaldehyde and then cry-protected in 20% sucrose. To detect effective knockdown of Daam2 and PIP5K in the chick, we generated mRNA probes and performed in situ hybridization (Lee and Deneen, 2012 (link)). For chick spinal cord immunohistochemistry, the following antibodies were used: mouse anti-Pax7 (DSHB), anti-Nkx2.2 (DSHB), anti-Myc (Sigma), and rat anti-HA (Sigma). Mouse spinal cord was analyzed using in situ hybridization: MBP, PLP, PDGFRα; and immunohistochemistry: chick anti-beta galactosidase (Abcam 1:1,000), mouse anti-MBP (Covance 1:500), mouse anti-PLP (1:500), rabbit anti-Olig2 (Abcam 1:1,000), rabbit anti-GFAP (Dako 1:1,000), rabbit anti-Iba-1 (Wako 1:1,000).

Spinal cords were fixed in 4% paraformaldehyde, and then cryoprotected in 20% sucrose. Mouse spinal cord was analyzed using colorimetric in situ hybridization as previously described (Kang et al., 2012 (link)). Immunohistochemistry: mouse anti-MBP (Covance 1:500), mouse anti-PLP (1:500), rabbit anti-Olig2 (Abcam 1:1000), rabbit anti-GFAP (DAKO 1:1000), rabbit anti-Iba1 (Wako 1:600).
To perform the two-color fluorescent in situ hybridization using the TSA-FITC/TSA-Cy5 system, we generated FITC labeled Apcdd1 probes, and combined with the probes of DIG labeled oligodendrocyte lineage markers (PDGFRα, PLP). Day 1 of the two-color fluorescent in situ hybridization was performed the same as for the colorimetric in situ. For day 2 of in situ, the slides were washed with 0.1X SSC and endogenous peroxidase activity was quenched by incubation in 3% H₂O₂ for 20 minutes. After quenching step, the slides were blocked with 0.5% of a blocking reagent containing TN (100 mM Tris-HCl, pH 7.5, 150 mM NaCl). The following antibodies were used to detect either FITC or DIG labeled probes; anti-FITC-POD, along with FITC-Tyramide; anti-DIG-POD with Cy5-Tyramide in Amplification Reagent (TSA^™ kit).