Alexa flour 488 goat anti rabbit igg antibody

At 24 h posttransfection, NIH 3T3 fibroblasts were plated on coverslips (7.5 × 10⁴ cells/well of a 12-well plate) and then fixed in 4% formaldehyde for 10 min. Cells were permeabilized with ice-cold methanol at −20°C for 20 min and incubated with rabbit polyclonal antiserum to ORF45 (generously provided by Ren Sun [54 (link)]; 1:200) in 5% BSA overnight at 4°C. Then Alexa Flour 488 goat antirabbit IgG antibody (Invitrogen) was added (1:1,000) for 1 h at room temperature. Coverslips were mounted in 4′,6-diamidino-2-phenylindole (DAPI)-containing Vectashield (VectorLabs). Imaging was performed using a confocal Zeiss LSM 880 NLO AxioExaminer microscope driven by Zen 2 software with a 40× water immersion objective (Zeiss and 1.0 NA). Image analysis was performed in FIJI, including image cropping, and brightness/contrast adjustments (103 (link)).

To detect P2Y6 receptor on membrane surfaces by FACS, the cells were fixed using 4% paraformaldehyde (Sigma-Aldrich) without permeabilization and were incubated and blocked using PBS containing 1% BSA for 30 min at room temperature. Next, the cells were labeled with the rabbit anti-P2Y6 receptor antibody (1:200; Alomone Labs) for 2 h at room temperature, followed by the Alexa Flour 488 goat anti-rabbit IgG antibody (Invitrogen) for 1 h at room temperature. Lysosomal activity was determined using Lyso-Tracker (Enzo Life Sciences) according to the manufacturer's protocol. The cells for flow cytometric analysis were washed three times with FACS buffer (PBS containing 1% FBS) and then analyzed using a FACSVerse flow cytometer (BD Biosciences). FlowJo software (Tree Star, OR, USA) was used for the data processing.

To detect the P2Y6 receptor on membrane surfaces, the cells were fixed using 4% paraformaldehyde (Sigma-Aldrich) and were incubated and blocked using PBS containing 1% BSA for 30 min at room temperature. Next, the cells were labeled with the rabbit anti-P2Y6 receptor antibody (1:200, APR-011; Alomone Labs, Jerusalem, Israel) for 2 h at room temperature, followed by the Alexa Flour 488 goat anti-rabbit IgG antibody (Invitrogen) for 1 h at room temperature. Lysosomal activity was determined using Lyso-Tracker (ENZ-51005, Enzo Life Sciences) according to the manufacturer's protocol. To determine the intracellular trafficking of the P2Y6 receptor, THP-1 cells were co-stained with anti-P2Y6 receptor and anti-Rab5 antibody (Santa Cruz Biotechnology) for early endosome marker. Finally, the cells were washed with 1% fetal bovine serum/PBS and mounted with 4′,6-diamidino-2-phenylindole-containing fluorescence microscopy mounting medium (Invitrogen). The samples for confocal analysis were analyzed using a laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).