Cy3 conjugated donkey anti mouse igg antibody

The cellular distributions of proteins were detected based on immunofluorescent staining technology [5 (link)]. For post-fixation, the brain sections or cell cultures were immersed into 100% cold methanol for half an hour. After blocking nonspecific binding site with 10% BSA/PBS, samples were incubated with primary antibodies (anti-EphA4, 1:100, Santa Cruz Biotechnology, Cat. No. sc-135897, RRID: AB_2099356; anti-MAP-2, 1:50, Cell Signaling Technology, Cat. No. 4542, RRID: AB_10693782; anti-NeuN, 1:200, Abcam, Cat. No. ab177487, RRID: AB_2532109) at 4 °C overnight. The samples were next incubated with appropriate secondary antibodies (CY3-conjugated donkey anti-mouse IgG antibody, 1:400, Jackson ImmunoResearch Laboratories, Cat. No. 715-165-150, RRID: AB_2340813; FITC-conjugated goat anti-rabbit IgG antibody, Cat. No. 111-095-003, RRID: AB_2337972) at 25 °C for 1 h. Then brain sections or cell cultures were washed, dried and mounted with a coverslip. At last, the results of immunofluorescent staining were observed with Olympus BX51 fluorescent microscope (Olympus, Japan).

Vero E6 cells grown on glass coverslips were infected with SARS-CoV-2 at an MOI of 1, in the presence or absence of 20 μM HK. After 16 h, cells were fixed with 3% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Coverslips were then rinsed 3× with PBS and permeabilized for 10 min with 0.2% Triton X-100 in PBS. Cells were incubated with primary antibodies in PBS with 5% FCS for 1 h at room temperature. Primary antibodies used were a mouse monoclonal anti-dsRNA J2 (45 (link)) and a rabbit anti-SARS-CoV nucleocapsid (46 (link)). Coverslips were washed and incubated with secondary antibodies: an Alexa488-conjugated goat anti-rabbit IgG antibody (ThermoFisher) and a Cy3-conjugated donkey anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories). Nuclei were stained with Hoechst 33258 (ThermoFisher). After mounting with Prolong Gold (ThermoFisher), coverslips were imaged using a Leica DM6B fluorescence microscope and LASX software.