Anti fade mounting medium containing dapi

Immunofluorescence was carried out for active caspase-3. Nonspecific binding was blocked for 1 h using 10% normal goat serum, 1% BSA, and 0.1% Triton X-100 and then incubated with rabbit antiactive caspase-3 primary antibody (1:900; R & D Systems) overnight at 4°C. A goat antirabbit FITC-conjugated secondary antibody was applied for 40 min at room temperature in the dark (1:200; Stratech Scientific, Newmarket, UK). Cover slips were mounted on glass slides with antifade mounting medium containing DAPI (Vector Laboratories) and assessed on a fluorescent microscope with a ×40 objective (Olympus, Southend-on-Sea, UK). Ten discrete glands each containing at least 20 cells were scored per animal from each treatment group. The total number of DAPI-stained nuclei and the total number of active caspase-3-positive cells were assessed per gland and presented as mean gland size and percentage of cells positive for active caspase-3. Data were normalized to untreated glands of each genotype.

Paraffin‐embedded brain sections were deparaffinized and rehydrated and subjected to antigen retrieval in sodium citrate buffer (pH 6.0) at 95°C for 10 min. After blocking, the sections were incubated with the primary antibody against Aβ at 4°C overnight. Biotin‐conjugated secondary antibody was added and incubated at 37°C for 30 min. Brown‐colored signals were generated by using the Vectastain ABC system (Vector Laboratories, Burlingame, CA, USA) and diaminobenzidine substrate (Wako). Finally, the sections were counterstained with hematoxylin and mounted for microscopy. Images were taken using an Olympus BX51 Light Microscope (Olympus, Tokyo, Japan). For tracing the GFP expression in brain tissues, paraffin‐embedded slides were deparaffinized, rehydrated, and mounted with Antifade Mounting Medium containing DAPI (Vector Laboratories) for 10 min, followed by the fluorescence imaging using an Olympus BX53 Fluorescence Microscope (Olympus).

Shrews (n = 5–6 shrews per group) were treated with either vehicle or yohimbine (1 mg/kg, i.p.) and rapidly anesthetized with isoflurane and subjected to perfusion at 15 min and 30 min post-treatment to examine 5-HT and SP immunoreactivity. The experimental procedure prior to staining was performed as described above for Section 4.4.1. c-fos Staining and Image Analysis. Coronal brainstem sections (20 μm) were blocked with 0.1 M PBS containing 10% donkey serum and 0.3% Triton X-100, then incubated overnight at 4 °C with a mix of goat anti-5-HT primary antibody (1:1000, ab66047, Abcam) and rat anti-SP primary antibody (1:400, MAB356, EMD Millipore, Burlington, VT, USA) in 0.1 M PBS containing 5% donkey serum and 0.3% Triton X-100. Sections were washed 3 times (10 min each) in PBS and incubated in a mix of Alexa Fluor 488 donkey anti-goat (1:500, ab150133, Abcam) and cy3-conjugated donkey anti-rat (1:500, AP189C, EMD Millipore) secondary antibody in 0.1 M PBS containing 0.3% Triton X-100 for 2 h at room temperature. After washing with PBS 3 times (10 min each), sections were mounted with anti-fade mounting medium containing DAPI (Vector Laboratories). Images for the DVC were acquired using a confocal microscope (Zeiss LMS 880) as described above. Fluorescence intensity (mean gray value) of 5-HT and SP values were acquired using ImageJ software, as described previously [45 (link)].