Rnascope 2.5 hd detection reagent kit red rnascope multiplex fluorescent reagent kit v2

Frozen brain slices with 14-µm thickness mounted on adhesion microscope slides (CITOGLAS) were subjected to in situ hybridization. The mouse Hrh2 probe and RNAscope 2.5 HD Detection Reagent Kit-Red/RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics) were used for Hrh2 expression in OL lineages, microglia, astrocytes, or pericytes, together with immunostaining of NG2, O4, CNP, CC-1, Olig2, Iba-1, GFAP, PDGFRβ, or GFP. RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics) was used for duplex hybridization by combining the Hrh2 probe C1 with a GFP probe C2 for validation of Hrh2 reexpression. For combined in situ hybridization and immunofluorescence analyses, the slices were then incubated with rabbit anti-NG2 (1:300, Millipore), mouse anti-O4 (1:150, Millipore), rabbit anti-CNP (1:400, Abcam), mouse anti-CC-1 (1:400, Abcam), rabbit anti-Olig2 (1:500, Millipore), rabbit anti-Iba-1 (1:200, Abcam), rabbit anti-GFAP (1:400, Lianke), goat anti-PDGFRβ (1:20, R&D Systems), or chicken anti-GFP (1:400, Abcam). Fluorescent images were taken using an Olympus FV1000 or Leica SP8 laser confocal microscope.