Animals were anesthetized with isoflurane, decapitated, and dissected. Tissue was fixed in RNase-free 4% PFA/PBS overnight at 4°C and equilibrated serially in 10% sucrose/PBS for >1 hr, 20% sucrose/PBS for >2 hr, and 30% sucrose/PBS overnight, all at 4°C. Tissue was then embedded in O.C.T. (TissueTek) and stored at –80°C. The tissue was sectioned at 10 μm using a Leica CM3050S cryostat and stored at –80°C.
Following the BaseScope protocol for fixed frozen sections, slides were baked for 50 min at 60°C and post-fixed with 10% neutral-buffered saline for 15 min at 60°C. This was followed by target retrieval for 5 min at 100°C and protease III treatment for 30 min at 40°C. Using the BaseScope Duplex Detection Reagent kit (Advanced Cell Diagnostics, 323810), subsequent steps of hybridization and detection followed the vendor’s protocol. Probes are listed inTable 2 . The probe set for Higd1c was custom-designed to target only the first two exons of Higd1c. For detection of Th mRNA, amplification steps 7 and 8 were reduced from 30 min and 15 min, respectively, to 15 min and 7.5 min for some samples. Images were collected on a Nikon Ti widefield inverted microscope using a DS-Ri2 color camera. Two sets of experiments were performed on tissues from C57BL/6J (2), Higd1c+/+ (3), and Higd1c-/- (2) animals (Figure 1D and E , Figure 1—figure supplement 4A–E,G–J ).
Following the BaseScope protocol for fixed frozen sections, slides were baked for 50 min at 60°C and post-fixed with 10% neutral-buffered saline for 15 min at 60°C. This was followed by target retrieval for 5 min at 100°C and protease III treatment for 30 min at 40°C. Using the BaseScope Duplex Detection Reagent kit (Advanced Cell Diagnostics, 323810), subsequent steps of hybridization and detection followed the vendor’s protocol. Probes are listed in