Dm550b

Skin biopsies were collected from the dorsal side of SPF and GF mice, fixed in 10% (w/v) formalin, embedded in paraffin, and sectioned at 6 μm. Tissue sections were stained with hematoxylin and eosin to characterize epidermal thickness or with toluidine blue to identify mast cells. For immunofluorescence, sections were deparaffinized with xylene and rehydrated in downgraded alcohol. Heat-inactivated antigen retrieval was performed by incubating the tissue sections in 10-mM sodium citrate buffer, pH 6.0, and subsequently washing the sections with a PBS/0.2% Triton solution. Tissue sections were blocked with 10% (v/v) normal goat serum for 2 h at room temperature. After blocking, sections were incubated with a primary antibody. The antibodies that were used include anti-mouse Keratin 6A (Biolegend), anti-mouse Loricrin (Biolegend), anti-mouse CD3 (Abcam), and anti-mouse Ki67 (Abcam). Following multiple washes, secondary antibodies, goat anti-rabbit IgG-Alexa, and goat anti-mouse-Alexa 555 were applied for 1 h at room temperature and then washed. Slides were mounted with prolong DAPI (Molecular Probes) and examined under a fluorescent microscope (Leica DM550B). Positive-stained cells were counted in five fields per tissue section at × 400 magnification, three tissue sections per mouse, and three mice per group.

Broad-band autofluorescence was acquired from sections cut at 3 μm using X20 objective (Leica DM550B). Sections were excited at 458 nm and fluorescence emission captured above 475 nm. Fluorescence intensity per airway epithelium was quantified using ImageJ software and divided by background emission. At least 10 small airways per mouse was analysed.

Ca_v1.2 fluorescent immunohistochemistry was performed to confirm elimination of Ca_v1.2. Fluorescent immunohistochemistry was also used to confirm injection placement. Mice were transcardially perfused with 4% PFA, and brains were dissected and postfixed overnight in 4% PFA followed by cryoprotection in 30% sucrose at 4°C for at least 72 h. Forty-micrometer-thick sections spanning the hippocampus were obtained using a sliding microtome and incubated in anti-chicken GFP (1:10,000, Aves Labs) and anti-rabbit glial fibrillary acidic protein (1:1000, Invitrogen) primary antibody overnight at 4°C. Sections were rinsed in 0.1 m phosphate-buffer (PB) and incubated with donkey AlexaFluor 488 (1:300) and AlexaFluor 568 (1:300) antibody for 1 h at room temperature. Doublecortin fluorescent immunohistochemistry was performed to analyze cells in the dentate gyrus that had recently committed to neuronal fate. Sections were incubated in anti-guinea pig doublecortin (1:5000, Millipore) primary antibody overnight at 4°C. Sections were rinsed in 0.1 m PB and incubated with donkey AlexaFluor 594 (1:400) antibody for 1 h at room temperature. Sections were imaged using an epifluorescent microscope (Leica DM550B with Leica Application Suite Advanced Fluorescence 3.0.0 build 8134 software, Leica Microsystems).

Fixation and sectioning were conducted in order to confirm injection targets and expression. Following the termination of behavioral testing, mice were anesthetized with an injection of euthasol (150 mg/kg, I.P.) and brains were fixed with a 4% paraformaldehyde via aortic arch perfusion followed by cryoprotection in 30% sucrose in 0.1 M 251 phosphate buffer (PB) at 4 °C for 72 h. Coronal sections were cut on a cryostat (20 μm). For cell body photometry tissue, GCaMP6s expression cells were identified via immunohistochemistry targeting GFP (primary—anti-GFP Abcam (1:200, ab13970); secondary—goat anti-chicken Alexa 488 (1:1000, Thermofisher, A-11039)). For DREADD experiments, hM4Di-expressing cells were identified via immunohistochemistry targeting mCherry (1:200, anti-mCherry Abcam (ab167453); secondary—goat anti-rabbit Alexa 488 (1:1000, Abcam, ab150077)). Sections were mounted using VECTASHIELD Antifade Mounting Medium with DAPI for fluorescent microscopy (Vector). Sections were imaged using an epifluorescent microscope 256 (Leica DM550B with Leica Application Suite Advanced Fluorescence 3.0.0 build 8134 257 software, Leica Microsystems).