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).
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).
