IFITM3 Clusters on Virus Containing Endosomes and Lysosomes Early in the Influenza A Infection of Human Airway Epithelial Cells

Kummer, Susann; Avinoam, Ori; Kräusslich, Hans-Georg

The cells were fixed using 4% paraformaldehyde (PFA) in 1 × phosphate buffered saline (PBS) for 10 min at 37 • C and subsequently treated with 0.05% Triton-X100 in PBS for 5 min. Blocking was performed using 5% BSA in PBS for 10 min. The following primary antibodies were used for immunofluorescence: mouse anti-NP (catalogue no. MAB8257, Merck, Darmstadt, Germany), rabbit anti-IFITM3 (catalogue no. 11714-1-AP, Proteintech, Manchester, United Kingdom), mouse anti-EEA1 (catalogue no. 610457, BD Transduction LaboratoriesTM, San Jose, CA, USA), mouse anti-Rab7 (catalogue no. ab50533, abcam, Berlin, Germany), mouse anti-Rab11 (catalogue no. sc-6565, Santa Cruz, Dallas, Texas, USA), and mouse anti-lamp1 (catalogue no. ab25630, abcam, Berlin, Germany). The cells were incubated with the indicated primary antibodies in a blocking solution for 1 h at room temperature, and washed three times in PBS. The fixation, cellular membrane permeabilisation, and blocking were repeated between the first and secondary antibody incubation, as described above. For STED microscopy, goat anti-mouse STAR RED (catalogue no. 2-0002-011-2, abberior, Göttingen, Germany) and goat anti-rabbit Atto594 (catalogue no. ABIN964988, antibodies-online GmbH) were used as the secondary antibodies. For the triple staining, including the non-diffracted 488 nm channel, donkey anti-goat Alexa 488 (catalogue no. ab150129, abcam, Berlin, Germany) was used. The secondary antibody staining was performed for 1 h at room temperature under exclusion of light. After repeated washing in PBS, cover slips were mounted on the objective slides using Mowiol.