Tritc conjugated goat anti rabbit igg

Mice were deeply anesthetized with an intraperitoneal injection of ketamine (100 mg per kg) and xylazine (15 mg per kg), and then perfused with 25 ml of 4% paraformaldehyde. Before slicing, brain was dissected out and post-fixed overnight in 4% paraformaldehyde. At the region of virus infection, coronal slices around 100 µm thick were cut on a vibratome by using Leica VT1000S. Fixed brain slices were rinsed with PBS three times and then permeabilized with 0.5% Triton X-100 and blocked with 10% normal goat serum. The slices were incubated with TOM20 (Rabbit, Santa Cruz, sc11415) overnight at 4 °C cold room. Next day, the slices were rinsed with PBS three times and then incubated with tetrarhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (Santa Cruz) in dark place for 1 h at room temperature. Slices were rinsed three times with PBS and then mounted between two glass coverslips in Vectashield (Vector Laboratories) and sealed with dental wax. The immunohistochemical staining slices were visualized using Zeiss LSM710 confocal microscope at 488 nm (Mito-GCaMP6f) and 568 nm (TRITC) excitation, and 490–550 nm and >560 nm emission, respectively.

The 20 (R)-Rg3 was provided by Dalian Fu Sheng Pharmaceutical Co. (Dalian, China). A solution of Rg3 as freshly prepared in DMEM (500 μg/ml) and filtered by 0.22-μm membranes. It was diluted with cell culture media to final concentration in different treatments. DMEM, fetal bovine serum (FBS), TRIzol and Lipofectamine™ 2000 reagents were purchased from Invitrogen (Camarillo, CA, USA). DMSO and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). The enhanced chemiluminescence (ECL) assay kit was purchased from Amersham (Pittsburgh, PA, USA). Anti-rabbit FUT4, PCNA, p-ERK1/2, ERK, β-actin, HRP-conjugated anti-mouse IgM, HRP-conjugated anti-rabbit and anti-mouse IgG antibodies were purchased from Proteintech group (Wuhan, China). EGFR and p-EGFR were purchased from Cell Signaling Technology (Boston, MA, USA). FITC conjugated goat anti-mouse IgG and TRITC conjugated goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-Ulex Europaeus (UEA) lectin was purchased by EY Laboratories (San Mateo, CA, USA), which preferentially recognizes the total fucose. Mouse anti-Giantin Golgi marker antibody and mouse anti-LeY antibody (BG-8) were purchased from Abcam (Cambridge, UK). AG1478 inhibitor was obtained from Sigma (St. Louis, MO, USA). Coomassie protein assay reagent was purchased from Bio-Rad (Hercules, CA, USA).