Alexa fluor 555 donkey anti mouse

hMOF-overexpressed OVCAR3 cells were seeded into plates containing glass coverslips and cultured for 24 h. The cell climbing slice was fixed with 4% paraformaldehyde for 2 h, permeabilized in 0.5% PBST (PBS containing 0.5% Triton X-100) for 10 min and blocked with 5% goat serum for 60 min. Then the cell slides were incubated with primary antibodies of hMOF (#ab200660, 1:1000, Abcam) and MDM2 (#66511-1-Ig, 1:500, Proteintech) at 4 °C overnight. After incubation with secondary antibodies (Alexa fluor 488 goat anti-rabbit, A0423, Beyotime, 1:1000; Alexa fluor 555 donkey anti-mouse, A0460, Beyotime, 1:1000) for 2 h at room temperature, the nuclei were counterstained with DAPI (1 μg/mL) for 5 min. The immunofluorescence images were captured by a Nikon NiE-A1 plus or Nikon C2+ confocal microscope, and analyzed using Nikon-elements AR software.

The paraffin-embedded brain was serially cut into 10 μm-thick sections in coronal plane. After the standard histological procedures, sections were incubated overnight at 4°C in the mixture of 1:100 rabbit anti-TLR2 (Boster Biotechnology Company, Wuhan, China)/1:100 mouse anti-NeuN (Millipore Corporation, Billerica, USA), 1:100 rabbit anti-TLR2/1:1,000 mouse anti-GFAP (Millipore Corporation, Billerica, USA), 1:100 rabbit anti-TLR2 (Boster Biotechnology Company, Wuhan, China), 1:100 rabbit anti-TLR4 (Boster Biotechnology Company, Wuhan, China)/1:100 mouse anti-NeuN (Millipore Corporation, Billerica, USA), 1:100 mouse anti-TLR4 (Abcam, Cambridge, USA)/1:1,000 rabbit anti-GFAP (Millipore Corporation, Billerica, USA), 1:100 mouse anti-TLR4 (Abcam, Cambridge, USA). Then the sections were incubated respectively with 1:800 Alexa Fluor 568 donkey anti-rabbit (Invitrogen Life Technologies, NY, USA), 1:300 Alexa Fluor 555 donkey anti-mouse (Beyotime) for one hour at room temperature, followed incubated with 1:800 Alexa Fluor 488 donkey anti-mouse (Invitrogen Life Technologies, NY, USA), 1:800 Alexa Fluor 488 donkey anti-rabbit (Invitrogen Life Technologies, NY, USA), 1:100 Lectin-FITC conjugate (Sigma-Aldrich, St. Louis, USA) for onehour at room temperature. Finally, these sections were observed under laser confocal microscope (Leica, Germany).