Cy3 conjugated goat anti rabbit igg antibody

Cultured NP cells were rinsed three times with PBS and fixed with 4% paraformaldehyde. After washing again with PBS, the cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and blocked with 5% FBS for 30 min. Thereafter, the NP cells were incubated overnight at 4 °C with antibodies against MMP-9 (1:100; Abcam) and then incubated with a Cy3-conjugated goat anti-rabbit IgG antibody (1:100; Boster Bio, Pleasanton, CA, USA) for 1 h at 37 °C. After washing in the dark, the cells were incubated with DAPI for 5 min. Cells were imaged using a fluorescence microscope (Olympus).

For 5-HT1AR and OX1R localization experiments, rat brains were fixed in 4% formaldehyde overnight, and then embedded in paraffin blocks and sectioned using a microtome. The hippocampal sections were incubated with goat anti-5-HT1AR (1:100; Abcam, Cambridge, UK) and rabbit anti-orexin receptor 1-ATTO-488 (1:60); Alomone Labs, Jerusalem, Israel) antibodies overnight at 4 °C. After washing with PBS, each section was incubated with a Cy3-conjugated goat anti-rabbit IgG antibody (1:100; Boster Biological Technology, Pleasanton, US) at 25 °C for 2 h. After a further wash with PBS, each section was incubated with DAPI (1:100,000; Invitrogen, California, US) at 25 °C for 10 min. The stained sections were examined under a Leica DMRE laser scanning confocal microscope (Leica, Milton Keynes, UK).

The lung sections were dewaxed, heated in the antigen retrieval reagent (18 mM citric acid and 82 mM sodium citrate) for 10 min and blocked with goat serum (Solarbio). The sections were incubated with anti-E-cadherin antibody (1:100, Cat. No.: BA0474, Boster, Wuhan, China) overnight at 4°C, followed by incubation with Cy3-conjugated goat anti-rabbit IgG antibody (1:200, Cat. No.: A0516, Beyotime) for 1 h at room temperature. Thereafter, the cell nuclei were briefly stained with DAPI (Biosharp, Korea). The sections were washed, mounted and observed under a BX53 fluorescence microscope (Olympus, Japan).