Protocol detail

In Situ Hybridization for Plp1 and Enpp6 Expression

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In situ hybridization using a proteolipid protein (Plp1) riboprobe was performed as previously described [55 (link), 56 (link)]. The Enpp6 plasmid vector (pCMV-SPORT6, gift from Dr. William D. Richardson, University College London) was used to generate the Enpp6 riboprobe (Xiao et al. 2016 [57 (link)]). In 15 μm coronal cryosections, hybridized Plp1 or Enpp6 riboprobe was detected with alkaline phosphatase-conjugated sheep anti-digoxigenin antibody and incubation in substrate solution (nitroblue tetrazolium chloride/5–bromo-4–chloro-3–indolyl-phosphate [NBT/BCIP]; Dako).

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Sullivan G.M., Knutsen A.K., Peruzzotti-Jametti L., Korotcov A., Bosomtwi A., Dardzinski B.J., Bernstock J.D., Rizzi S., Edenhofer F., Pluchino S, & Armstrong R.C. (2020). Transplantation of induced neural stem cells (iNSCs) into chronically demyelinated corpus callosum ameliorates motor deficits. Acta Neuropathologica Communications, 8, 84.

Publication 2020

NBT/BCIP

Alkaline phosphatase Anti antibody Cryosections Digoxigenin In situ hybridization Nitroblue tetrazolium chloride Phosphate Plasmid Protein Proteolipid Sheep Vector

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Variable analysis

independent variables

Type of riboprobe used (Plp1 or Enpp6)

dependent variables

Presence and localization of Plp1 or Enpp6 mRNA expression detected by in situ hybridization

control variables

Cryosection thickness (15 μm)
Use of alkaline phosphatase-conjugated sheep anti-digoxigenin antibody
Incubation in substrate solution (NBT/BCIP)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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