S4030

The primary probe staining procedure was similar to those in previous reports^{27 (link)–29 (link)}. Tissue sections were stained with oligo-conjugated WGA in 1× hank’s balanced salt solution (HBSS) buffer for 20 min at 37 °C. Samples were post-fixed in 4% PFA for 10 min. Tissue sections were permeabilized using 0.5% Triton in DPBS for 20 min at room temperature before primary probe staining. Tissue samples were incubated for 5 min in pre-hybridization buffer composed of 50% (vol/vol) formamide and 2 mM Ribonucleoside vanadyl complexes (Sigma-Aldrich, R3380) in 2× saline sodium citrate (2× SSC) (Invitrogen, AM9765). Tissue samples were then stained with primary probes in primary hybridization buffer, containing 24-28 μM primary probes, 50% (vol/vol) formamide, 0.1% yeast tRNA (Invitrogen, 1885325), 1% (vol/vol) murine RNase inhibitor (NEB, M0314S), 10% (wt/vol) dextran sulfate (Millipore, S4030), and 2 μM anchor probe (a 15-nt sequence of alternating dT and thymidine-locked nucleic acid with a 5′ -acrydite modification) in 2× SSC, in a humidity chamber at 37 °C for 24 h. After staining, samples were washed for 15 min with 2× SSCT (2× SSC, 0.1% (vol/vol) Tween 20) twice at 60 °C and then once for 15 min at room temperature.