Alexfluor 488 secondary antibodies

Epithelial cells were cultured on glass coverslips in 24-well plates, infected with clinical EPEC isolates, washed with PBS and fixed using either cold methanol (5 min) or 3.7% paraformaldehyde in PBS (20 min), washed with PBS, then permeabilized with 0.1% Triton X-100 (5 min) at room temperature and incubated overnight in blocking solution (Invitrogen, 000–105). Cells were labeled with anti-ZO-1 (Invitrogen 617300) in a 1:100 dilution, mouse anti-occludin (Invitrogen 33–1500) at a 1:100 dilution at 4°C overnight, or 1 U of BODIPY-558/568 Phalloidin (Invitrogen, B3475) diluted into 50 µL of blocking solution for 1 h. Cells were incubated with AlexFluor-488 secondary antibodies (Invitrogen, A11034 or A11029) at 1:250 in Invitrogen blocking solution for 2 h at room temperature, nuclei were stained with Hoecsht 33342 (Invitrogen, H3570) and coverslips mounted using ProLong Gold Antifade reagent (Invitrogen, P36934). Images were acquired using either a Leica DMI4000 (MetaMorph software) fluorescence microscope, or a Leica TCS SPE DMI 4000B (LAS X software) confocal microscope and processed with ImageJ and Adobe Photoshop 2020.

The experiments were performed as described previously [25 (link)]. Yeast cells were grown to mid-log phase, shifted for 1 h to their restrictive temperatures and fixed with 4% formaldehyde for 1 h at room temperature. After zymolyase treatment, spheroplasts were transferred to polylysine-coated microscope slides, permeabilized with 0.5% triton X-100/P-solution (0.1 M phosphate-buffer pH6.5, 1.2 M sorbitol), blocked with antibody blocking buffer ABB (1x PBS, 10% heat-inactivated FCS, 0.3% tween-20) for 1 h at room temperature and incubated with anti-Nop1 antibodies diluted 1:2500 in ABB overnight at 4°C. Following four times washing with ABB, anti-rabbit Alexa Flour 594 secondary antibody (from Invitrogen) diluted 1:100 in ABB was added for 2 h at room temperature. After washing with ABB and 0.1% Tween-20/PBS, the DNA was stained with Hoechst 33342 and microscopy studies were performed as described in “Fluorescence in situ hybridizations”.
For co-localization of 25S rRNAs and Nop1 proteins, the in situ hybridization protocol was performed first with the Cy3-labeled oligonucleotide HK2200 against 25S rRNAs followed by the immunofluorescence protocol with anti-Nop1 primary antibodies and anti-rabbit Alex Fluor 488 secondary antibodies (from Invitrogen).