Ab66047

Mice were transcardially perfused with 4% paraformaldehyde and then postfixed overnight. Coronal brain slices containing the LC or dorsal raphe were cut at a thickness of 50 μm, using a vibratome (Leica VT1000S). Free-floating slices were permeabilized with 0.5% Triton-X 100 and blocked with 1% fish gelatin in 0.1 M phosphate-buffered saline (pH 7.4). NE-positive neurons were immunostained by incubating overnight with rabbit anti-dopamine beta-hydroxylase (1:500, Abcam ab209487) and labeled with Alexa 546-conjugated donkey anti-rabbit secondary antibody (1:1000, Invitrogen). 5-HT neurons were immunostained by incubating with goat anti-5-HT antibody (1:500, Abcam ab66047) and similarly labeled with Alexa 647-conjugated donkey anti-goat secondary antibody (1:1000, Invitrogen).

For cryoprotection, brains were gradually immersed in 10, 20, and 30% sucrose in 24 h intervals and embedded in OCT compound (Leica Microsystems, Bensheim, Germany) with liquid nitrogen. The brains were cut into frozen coronal sections (30 μm) using a Leica CM3050 cryostat and then stored in a free-floating buffer. For immunohistological analysis, the sections were blocked in 5% normal chicken serum (containing 0.3% Triton X-100) for 1 h. After washing, the sections were incubated with primary antibodies against ionized calcium-binding adaptor molecule 1 (Iba-1, 1:200, 019-19741, Wako) or 5-hydroxytryptamine (5-HT, 1:200, ab66047, Abcam) overnight at 4°C. Subsequently, the sections were incubated with goat anti-rabbit IgG HRP (1:400, ab6722, Abcam) or Alexa Fluor^® 488 donkey anti-goat IgG H&L (1:400, ab150129, Abcam) secondary antibodies for 2 h at RT. To analyze the Iba-1-positive cells, the sections were incubated with an avidin-biotin-peroxidase complex (VECTASTAIN ABC kit, Vector Laboratories) for 2 h. The peroxidase activity was visualized using a stable diaminobenzidine solution. To analyze the 5-HT-positive signal, the sections were exposed to DAPI (1:1,000, D9542, Sigma) to stain cell nuclei. Immunoreactivity was observed under an Axio-Phot microscope (Carl Zeiss, Germany). The intensity was quantified using ImageJ 1.46 software (NIH, Bethesda, MD, USA).

The protein activities of tryptophan hydroxylase 2 (TPH2), 5-HT, BDNF, CREB, and p-CREB proteins in the hippocampus were evaluated by western blotting, as described by Grønli et al. (Grønli et al., 2006 (link)). The proteins from homogenates were separated by 10% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 5% skim milk, the membranes were probed overnight at 4°C with primary antibodies: Rabbit anti-TPH2 monoclonal antibody (1:1,000, ab 111828, Abcam), Goat anti-5-HT monoclonal antibody (1:1,000, ab66047, Abcam), Rabbit anti-CREB monoclonal antibody (1:1,000, ab32515, Abcam), Rabbit anti-CREB (phospho S133) monoclonal antibody (1:1,000, ab32096, Abcam), Rabbit anti-BDNF monoclonal antibody (1:1,000, ab108319, Abcam), and Goat anti-beta actin polyclonal antibody (1:1,000, ab8229, Abcam). The membranes were washed and incubated for 2 h with HRP-conjugated anti-rabbit secondary antibodies. Western blots were visualized using an enhanced chemiluminescence (ECL) advanced kit. The protein expression was semi-quantified using ImageJ (NIH).

Immunofluorescence analyses were performed using a modified method described by the previous studies (Zhang et al., 2016 (link); Zhao et al., 2017 (link)) to observe the 5-HT and GR relative intensity in dorsal raphe nuclei (DRN) and the hippocampal CA1 area, respectively. Brain tissues were cryoprotected in 30% sucrose, embedded in tissue-freezing medium with liquid nitrogen, and cut into frozen coronal sections (35 μm) using a Leica CM3050 cryostat. Sections were stored under anti-freeze buffer. Parallel free-floating sections were treated with blocking buffer (5% normal chicken serum in PBS and 0.3% Triton X-100 for 1 h at 4°C) and incubated with primary antibodies against 5-HT (1:400, ab66047, Abcam) or GR (1:200, sc-393232, Santa Cruz) overnight at 4°C. After washing with ice-cold PBS, sections were incubated with donkey anti-goat IgG H&L (1:400, Alexa Fluor^® 488, ab150129, Abcam) or goat anti-mouse IgG H&L (1:400, Alexa Fluor^® 594, ab150116, Abcam) secondary antibodies for 2 h at 4°C. The sections were subsequently exposed to DAPI (1:1,000, D9542, Sigma) to stain cell nuclei. All immunoreactions were observed under an Axiophot microscope (Carl Zeiss, Germany). Total signal intensity was quantified using the ImageJ 1.46 version (NIH, Bethesda, MD, USA) and compared relatively with the control group.

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)].

The slices were fixed at room temperature for 30 min, washed six times 1x PBS for 5 min each, and then placed in 1% BSA blocking solution at room temperature for 60 min. The blocking solution was sucked up, and anti-GB1 antibody (1:1000, Abcam, USA, ab55051) and anti-5-HT antibody (1:1200, Abcam, USA, ab66047) were added to incubate for 1 h at room temperature and then overnight at 4°C. After washing three times in 1x PBS, the slices were incubated in the dark with secondary antibodies (rabbit anti-mouse IgG, 1:300, Abcam, USA, ab150125; rabbit anti-goat IgG, 1:300, Abcam, USA, ab6738) at room temperature for 2 h. The slices were washed again three times, placed on the slide and drop the anti-fade fluorescence mounting medium (Thermo Fisher Scientific, USA), and covered with the slide. After air-drying in the dark, the slides were observed using a confocal microscope (Zeiss, Germany).