Er staining kit red fluorescence cytopainter

48 hours after transfection, cells were fixed with 4% paraformaldehyde/PBS solution for 10 min. at room temperature, washed with PBS, and permeabilized in 0.1% TritonX-100/PBS for 1 min. Cells were incubated with 1 μM of SNAP Surface Alexa Fluor 488 (New England BioLabs #S9129S, Ipswich, MA) and 1:1000 ER Staining Kit-Red Fluorescence-Cytopainter (Abcam #139482, Cambridge, MA) at 37 °C for 30 min. protected from light. Hoechst 33342 was used for nuclear staining. Cover slips were mounted using ProLong Glass antifade reagent (Thermo Fisher #P36982). Confocal fluorescence microscopy was performed using Leica SP8X laser scanning confocal microscope equipped with a 40x oil immersion objective (Leica Camera, Wetzlar, Germany). The detection pinhole was set to 1 Airy unit, light collection configuration was optimized according to the combination of chosen fluorochromes (Alexa Fluor 488, Texas Red, and Hoechst), and sequential channel acquisition was performed to minimize the risk of bleed-through. The intensity gain was adjusted for each channel before capture in order to avoid saturated pixels. 8 bit, 1024 × 1024 pixel images were collected as Z-stack acquisition. All microscopy was performed in collaboration with the W.M. Keck Microscopy center on the University of Washington School of Medicine campus.

DAPI stock solution of 1 mg/mL (Sigma, Rehovot, Israel), ER staining kit Red Fluorescence Cytopainter (Cat. 139482, Abcam, Cambridge, MA, USA), Concanavalin A Alexa fluor 594 Conjugate (Cat. 11253, Invitrogen, Molecular probes, Darmstadt, Germany). Stock solutions were prepared according to manufactures protocol.

Cells were seeded on 4-well slides and transduced with retrovirus. 2 days after transduction, cells were fixed with 4% paraformaldehyde, permeabilized with 1% Tween-20 and blocked with 1% BSA. The cells were incubated with the primary antibody against V5 (Invitrogen) overnight at 4 °C followed by incubation with biotinylated goat anti-mouse IgG antibody (H + L) (Vector Laboratories, BA-9200). Cells were subsequently labeled with AMCA using Fluorescent Streptavidin Kit (Vector Laboratories; AS-1200). For endoplasmic reticulum staining, ER Staining Kit-Red Fluorescence-Cytopainter (Abcam, Cambridge, MA; ab139482) was applied according to the manufacturer’s instruction. The slides were mounted using ProLong Gold Antifade Reagent (Invitrogen), and the cells were examined by LSM 880 confocal laser scanning microscope (Carl Zeiss, Germany).