Ab420

To visualize the localization of Nuc1-HA protein and tRNA-Gly in the fungal cells, immunocytochemistry and in situ hybridization were simultaneously performed. Sporidia cells of U. maydis strain expressing Nuc1-HA were fixed with 3% formaldehyde in phosphate-buffered saline (PBS). To permeabilize, cells were incubated with 0.1% Triton-X100 for 10 min. The cells were hybridized with DIG-labeled DNA probe for tRNA-Gly at 42°C overnight. After washing, cells were incubated with rabbit anti-HA antibody (H6908; Sigma-Aldrich; 1: 500 dilution) and mouse anti-DIG antibody (ab420; Abcam, Cambridge, UK; 1: 500 dilution) in TBS-T containing 5% skim milk for 1 h at room temperature. After washing, cells were incubated in TBS-T containing goat anti-Rabbit IgG cross-adsorbed secondary antibody conjugated with Alexa Fluor 488 (R37116; Thermo Fisher Scientific) and goat anti-mouse IgG Cy3 conjugate (AP181C; Merck, Darmstadt, Germany; 1: 500 dilution) for 1 h at room temperature. DAPI staining was performed at final concentration 1 μg/ml for 5 min at room temperature. After washing with TBS-T, the samples were analyzed using a Zeiss Axio Observer 7 microscope equipped with Colibri 7 under filter set 38HE for Alexa 488, 45 for Cy3 and 96HE for DAPI fluorescence (Carl Zeiss, Oberkochen, Germany).

To visualize mRNA in red color, after fixation HeLa cells were incubated with oligo-dT-[Cy3], diluted in SSC 2X, 1 mg/ml yeast tRNA, 0.005% BSA, 10% dextran sulfate, 25% formamide, for 2 h at 37 °C. Wash steps were performed using 4X and then 2X SSC buffer (0.88% sodium citrate, 1.75% NaCl, pH 7.0). To visualize mRNA in blue color for SGs experiments, the oligo-dT with digoxigenin was used after cells fixation with the same incubation procedure as oligo-dT-Cy3. Then the primary anti-digoxigenin antibodies (mouse, ab420, Abcam) and secondary antibodies (goat anti-mouse, Alexa 350, Invitrogen) were applied to cells according to supplier’s protocol.

PD-L1 was stained in parallel with oxRADD. The oxRADD protocol was completed as described above. After the 1 h incubation with the gap filling mixture, the slides were washed three times in PBS and blocked in 5% normal goat serum (Thermo Fisher 31873) in PBS for 30 min. Anti-Digoxigenin (Dig) antibody (abcam; #ab420 clone 21H8) at 1:250 and anti-PD-L1 antibody (abcam #ab205921 clone 28−8) at 1:500 was incubated in 5% normal goat serum in PBS at 4 °C overnight. The next day slides were washed three times in PBS for 5 min each, and the Alexa Fluor 546 goat anti-mouse secondary and Alexa Fluor 647 goat anti-rabbit secondary (Invitrogen) were incubated at a dilution of 1:400 in 2% BSA in PBS for 1 h at RT. Hoechst 33342 was added at a final dilution of 1:1000 for 15 min at RT to stain the nuclei. Slides were washed three times in PBS for 5 min each, briefly dried, and mounted with coverslips using ProLong Gold Antifade reagent. Slides were allowed to dry overnight in the dark at RT and visualized using a Nikon A1R confocal microscope or stored at 4 °C until analysis.