Ab209487

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).

Another 12 mice per group were deeply anesthetized by 8% chloral hydrate, followed by ventricular perfusion with normal saline and 4% paraformaldehyde solution in 0.1 M PBS. Frozen brain blocks were coronally sectioned to 20 μm using a freezing microtome (Leica CM1900, Germany). After rinse with PBST (0.3% Triton-100 in 0.01 M PBS), sections were blocked by 10% goat serum for 30 min and then incubated with primary antibodies, dopamine beta-hydroxylase (DβH, ab209487, Abcam, 1:1,000), serotonin transporter (SERT, AB9726, Millipore, 1:1000) and tyrosine hydroxylase (TH, AB152, Millipore, 1:2000) for overnight at 4°C. Subsequently, the second antibody fluorophore-conjugated donkey anti-rabbit IgG H&L (Alexa flour 555, Invitrogen, 1:500) was added to incubate for 2 h at room temperature. After three times rinse, the sections were mounted with 70% glycerol. Stereological quantification was performed as previously described in our laboratory (Zhang et al., 2015 (link)). The number of DβH-, SERT- and TH-immunoreactive (ir) neurons was counted by optical fractionator unbiased stereological method using a fluorescent microscope (Zeiss, Germany) with Stereo Investigator (Micro Bright Field, USA).

Immunofluorescence analysis of murine tumor mass was performed as follows. Samples were rapidly excised, fixed in buffered 4% formaldehyde for 24 h and paraffin-embedded. Histological sections, 5 μm thick, were cut from samples, were deparaffinized and boiled for 10 min in sodium citrate buffer (10 mM, pH 6.0; Bio-Optica) for antigen retrieval and immunostained over night at 4 °C with primary antibodies (anti-PD-L1, ab238697, Abcam; anti-β3-AR, ab94506, Abcam; anti-CD4, 14-9766-82, eBioscience; anti-CD8, 14-0808-82, eBioscience; DBH, ab209487, Abcam). Immune reaction was revealed incubating sections with secondary antibodies Alexa Fluor 488-conjugated IgG or Alexa Fluor 594-conjugated IgG (1:350; Jackson Laboratory). After counterstaining with 4,6-diamidino-2-phenylindole (DAPI), representative images were acquired by an Olympus BX63 microscope coupled to CellSens Dimension Imaging Software version 1.6 (Olympus). The immunofluorescence staining was evaluated through the ImageJ software (NIH, USA) as the fluorescence intensity of protein of interest was normalized to DAPI values.