2.5 hd assay brown

For in situ hybridization (ISH), adult STAT1^LepRKO, STAT3^LepRKO, STAT1STAT3^LepRKO and control mice were anesthetized with isoflurane and then euthanized by decapitation. Whole brains were dissected, flash frozen in isopentane, chilled on dry ice, and stored at −80 °C. 16 μm-thick coronal sections were cut on a cryostat (Leica), thaw-mounted to SuperFrost Plus slides, allowed to dry at −20 °C for one hour and then stored at −80 °C. Slides were then processed for ISH using RNAScope technology per the manufacturer's protocol (Advanced Cell Diagnostics) using either the 2.5 HD Assay-Brown (322310) or the multiplex fluorescent assay (320850) and Stat1 (492731) and Cre (312281-C3) probes. Images of the colorimetric assay were obtained with an Olympus BX51 and Olympus DP80 color camera under 40X objective with a 0.63X C-Mount for a total effective magnification of 25.2X. Fluorescent images were obtained with an Olympus BX53 and QImaging Retiga 6000 monochrome camera under 20X objective. Photoshop (Adobe) was used to adjust white balance (for color images) and adjust levels to remove nonspecific background (for each channel of the fluorescent images). All images from an experiment were processed identically.