Alexa fluor 555 conjugated goat anti rabbit igg

Immunofluorescence was used to assess the in-situ expression of the FRY. MDA-MB-231 related cells were cultured on coverslips in DMEM medium with 10% FBS and penicillin/streptomycin at 37°C with 5% CO₂ for 24 h. The cells were briefly washed in PBS, fixed with 4% formaldehyde for 30 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min and incubated in blocking buffer (10% goat serum, 0.2% Triton X-100, 0.05% Tween-20 in PBS, pH 7.4) for 1 h at room temperature. The cells were then incubated with primary antibodies diluted in blocking buffer overnight at 4°C. After rinsing, the primary antibodies were detected with Alexa Fluor® 594 conjugated goat anti-mouse IgG (Cell Signaling Technology) or Alexa Fluor® 555 conjugated goat anti-rabbit IgG (Cell Signaling Technology) secondary antibodies diluted at 1:500 in blocking buffer for 1 h at room temperature. Coverslips were mounted using ProLong® Diamond Antifade Mountant with DAPI (Life Technologies) and fluorescence images were visualized and captured with a ZEISS Axio Observer inverted fluorescence microscope (ZEISS, Jena, Germany).
Primary antibodies and dilution used in applications include the following: mouse anti-FRY (2F2-D8-E3-G3) antibody (RayBiotech, 1:100), mouse monoclonal anti-β-Actin antibody (Sigma-Aldrich, 1:2,000).

Immunostaining was performed as described previously [34 (link), 37 (link), 44 (link)]. In brief, tissue samples were sectioned (15 μm thickness) using a cryostat (CM1950, Leica Microsystems). Tissue sections were treated with 0.15% Triton X-100, blocked with 5% normal goat serum, and probed with primary antibodies against TAAR1 (rabbit, 1:300, Thermo Fisher Scientific, catalog no. PA5-115999), NeuN (mouse, 1:600, Cell Signaling Technology, catalog no. 94403s), GS (mouse, 1:1000, Abcam, catalog no. ab64613), CGRP (mouse, 1:500, Abcam, catalog no. ab81887) and NF200 (mouse, 1:500, Abcam, catalog no. ab215903). TG sections were subsequently visualized with Alexa Fluor 555-conjugated goat anti-rabbit IgG (1:300, Cell Signaling Technology, catalog no. 4413s), DyLight 488-conjugated goat anti-mouse IgG (1:300, Cell Signaling Technology, catalog no. 4408s), or IB4-fluorescein isothiocyanate (5 µg/ml; Sigma–Aldrich, catalog no. L2895). Antibodies are validated by the manufactures, and can be referred to datasheets of respective antibodies, which are listed in the Supplementary materials (Table S1). Images were acquired under an upright fluorescence microscope (Nikon 104 C) with a CoolSnap HQ2 CCD camera (Photometrics).