Tsa fluorescein

Briefly, 2 mm biopsies were performed on lesional and nonlesional skin as part of punch grafting treatment for 3 patients. Control skin was acquired from tumor excision tips without notable pathology from patients without vitiligo. Skin samples were immediately frozen and embedded in OCT. Tissues were stored at –80°C, and cryosections (10 mm thick) of skin were collected on Fisherbrand Superfrost Plus microscope slides. Sections were dried for 60–120 minutes at –20°C then used immediately or within 10 days. In situ hybridization was performed according to the RNAscope Multiplex Fluorescent Reagent Kit v2 (catalog 320293).
Briefly, slides were fixed in cold 4% PFA for 15 minutes and were then dehydrated in 50%, 70%, and 100% ethanol for 5 minutes each at room temperature (RT). H₂O₂ was applied for 10 minutes at RT and treated with protease IV for 30 minutes. C2 and C3 probes were diluted in C1 probes at a 1:50 ratio and incubated for 2 hours at 40°C. C1 probes were detected with TSA-fluorescein (Akoya Biosciences), C2 probes with Opal-620, and C3 probes with Opal-690 (Akoya Biosciences). Before mounting, DAPI was added to label the nuclei. Images were acquired using a Leica SP8 FALCON/DIVE (20× objective, 0.75 NA).

To investigate the coexpression of npy and agrp or cart and pomc in the tuberal hypothalamus, fluorescence double labeling ISH was done as described in Eilertsen et al. (2018 (link)), and replacing 50% deionized formamide with 4 M urea. DIG‐labeled riboprobes were incubated with sheep polyclonal anti‐DIG antibody (anti‐digoxigenin‐alkaline phosphatase FAB‐fragment, 1:2000, cat. # 11093274910, Roche Diagnostics, RRID: AB_514497) and detected with either Fast Red tablet (Roche Diagnostics) dissolved in 0.1 M Tris‐HCl pH 8.2 and 0.1% Tween‐20 or with 100 mg/ml Fast Blue BB salt (MilliporeSigma) and 100 mg/ml naphthol AS‐MX phosphate (MilliporeSigma) in 0.1 M Tris‐HCl pH 8.2, 50 mM MgCl₂, 0.1 M NaCl, and 0.1% Tween‐20 (MilliporeSigma). A 2% blocking solution (MilliporeSigma) in 2× saline‐sodium citrate buffer was used for blocking the sections, followed by the visualization of FITC‐labeled riboprobes using sheep polyclonal anti‐FITC (anti‐fluorescein conjugated with horseradish peroxidase, Fab fragments, cat. # 1142636910, Roche, RRID: AB_840257) and TSA™ Fluorescein (Akoya Biosciences Marlborough, USA) according to the manufacture's protocol. Sections were mounted with ProLong Glass antifade medium with NucBlue (Invitrogen).

RNA in situ hybridization (ISH) was performed with RNAscope technology (Advanced Cell Diagnostics). The probes to detect Ly6a, Clu, mKi67, and Mdm2 mRNA were MmLy6a-427571, MmCLuC3-427891-C3 and Mmki67C2-416771 and MmMdm2C2-447641-C2, respectively. For chromogenic assay, ISH was conducted using RNAscope 2.5 HD Assay-BROWN as per the manufacturer’s protocol. For fluorescent assay, RNAscope Multiplex Fluorescent V2 Assay was used. Either the Opal Fluorophore reagent packs (Opal 520, Opal 570, Opal 690) (Akoya Biosciences) or the tyramide signal amplification (TSA) fluorophores (TSA Fluorescein, TSA Cyanine 3, TSA Cyanine 5) (Akoya Biosciences) were used for detection. Images were acquired using a Nikon Ti2 inverted confocal microscope platform.