To prepare tissue sections for smFISH, mouse brains were dissected, immersed in 4% PFA for 4–6 h, and then dehydrated with 20–30% DEPC-treated sucrose for 24 h. Subsequently, the tissues were rapidly frozen on dry ice, embedded in O.C.T. compound, cryosectioned at a thickness of 20 µm, and mounted onto SuperFrost Plus microscope slides. The probes targeting against Rax (19046 A), Scn7a (18199B), and Col25a1 (19032A) were designed and validated by Advanced Cell Diagnostics. RNAscope v2 Assay (Advanced Cell Diagnostics, #320511) was used for all smFISH experiments according to the manufacturer’s protocol53. Briefly, the brain sections were dried at 55 °C for 2 h, rinsed with 1×PBS, treated with 3% hydrogen peroxide in methanol, and subjected to antigen retrieval. Subsequently, the tissue sections were dehydrated with 100% ethanol and incubated with mRNA probes for 2 h at 40 °C. The specific signals were then amplified with multiplexed amplification buffer and detected with TSA Plus fluorophore (Perkin Elmer, #NEL753001KT) for 30 min. To combine smFISH with the pulse-chase assay, we further treated the tissue sections with 0.5% Triton-X-100 and EdU detection solution. After staining, the slides were mounted and imaged for further analysis.

To detect the mRNA expression level of Mia, we used the hybridization chain reaction (HCR) approach54. We designed the probe sequences (Supplementary Table 2) using the coding and 3′UTR regions of Mia and synthesized the probes in Sangon Biotech, China. The brain sections were permeabilized in 70% ethanol for 16 h at 4 °C, followed by 0.5% Triton X-100 in 1×PBS at 37 °C for 1 h, and treated with 10 µg/mL Protease K was to improve mRNA accessibility. After two washes with 1×PBS at room temperature, sections were pre-hybridized in probe hybridization buffer (30% formamide, 5×SSC, 9 mM citric acid, 0.1% tween 20, 50 µg/mL heparin, 1×Denhardt’s solution, and 10% dextran sulfate) for 1 h at 37 °C and then incubated in probe hybridization buffer containing HCR probes (10 µM for each) at 37 °C for 3 h. After mRNA hybridization, the washing and amplification steps were performed as previously described54.

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