Alexa 488 conjugated goat anti rabbit igg antibody

In all, 8–12-week-old Cx3cr1Cre^+/− mice or E18.5-tamoxifen-labeled Cx3cr1Cre^+/−/R26Tdt^+/− mice were euthanized by carbon dioxide (CO₂) and perfused with PBS. Kidneys were excised and fixed in 4% paraformaldehyde overnight. After washing with PBS, kidneys were immersed in 30% sucrose overnight, snap frozen in optimal cutting temperature (OCT) compound and sectioned (10 µm). Slides were immunostained with 1:400 dilution of rabbit anti-GFP antibody (A-11122, Invitrogen) overnight at 4 °C, and visualized with 1:500 dilution of Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen). Images were assessed and acquired at ×10 or ×20 magnification on a Leica DMRE Research Epi-Fluorescence microscope. In all, 2–4 views (×20) were randomly acquired from the cortex or medulla and the average cell counts were quantified by individual who is blinded to the experimental design.

ACE2-GFP transfected HEK293T cell at 70% confluent were washed with PBS, fixed for 10 min with cold 100% methanol, and blocked using 5% BSA in PBS for 2 h. Subsequently, cells were incubated with a 1:2000 diluted anti-ACE2 rabbit polyclonal (10108-T24, Sino Biological) overnight at 4 °C, washed with PBS and incubated for 2 h with Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen). After staining, slides were mounted using ProLong Antifade (Thermo Fisher) solution and observed using a Leica SP8 confocal microscope.

Cryosections of 8 μm thickness were cut from the TA and SOL muscles. Immunohistochemistry was performed as described previously [26 (link)]. Sections were stained with polyclonal rabbit antibodies directed against connexin 43 (abcam), and Alexa 488-conjugated goat anti-rabbit IgG antibody (Invitrogen) was used as a secondary antibody. Fluorescence images were obtained using a BZ-9000 fluorescence microscope (KEYENCE).

SeV (strain Cantrell, clone cCdi; GenBank accession number: AB855654) was prepared as previously described [39 (link)] and kindly provided by Dr. Takashi Irie (Hiroshima University, Japan). Briefly, LLC-MK2 cells were infected with SeV and the supernatant was collected and used without purification. To titrate viral infectivity, prepared virus was diluted 10-fold in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, cat. D6046-500ML) containing 10% FCS, and LLC-MK2 cells were infected with dilutions of the virus at 37°C. At 1 hour postinfection, the cells were washed with phosphate-buffered saline (PBS) and cultured with DMEM containing 10% FCS at 37°C. At 1 day postinfection, the infected cells were fixed with acetone (Nacalai Tesque, cat. 21914–03)/methanol (Nacalai Tesque, cat. 00310–95). To calculate the viral titer (cell infectious unit [CIU]), the fixed cells were stained with a rabbit anti-SeV polyclonal antibody [40 (link)] as the primary antibody and an Alexa 488–conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific, cat. A-11008) as the secondary antibody, and the number of fluorescent foci per well was counted.