A21206

A549 cells were infected at an empirically determined MOI (by titration) that gave ~10% levels of infection for 6 hr, after which cells were fixed, permeablised and stained for a range of viral proteins. M2 was detected by ab5416 (Abcam, Cambridge, UK), NS1 by an in-house (rabbit) antiserum, NA by an in house (mouse) antiserum, NP by ab20343 (Abcam, Cambridge, UK) and PB2 by an in house rabbit antiserum. Secondary antibodies were species specific Alexafluor-488 or Alexafluor-546 (A21202, A11005, A21207 and A21206, Life Technologies, Loughborough, UK).

Sections (4 μm thick) from the paraffin-embedded colon tissue blocks were prepared for IHC or IF staining. Briefly, staining was performed with primary antibodies against TNF-α (1 : 600, rabbit polyclonal, Bioss, bs-2081R), IL-1β (1 : 2,000, rabbit polyclonal, ABclonal, A11369), NF-κB/p65 (1 : 5,000, rabbit polyclonal, Servicebio, GB11142), F4/80 (1 : 1000, CST, 70076), CD86 (1 : 500, Bioss, bs-1035R), and CD163 (1 : 300, Bioss, bs-2527R). Goat anti-rabbit IgG (HRP) (1 : 4,000, Abcam, ab205718), Alexa 488-conjugated donkey anti-rabbit IgG(H+L) (1 : 400, Life Technologies, A21206), and Cy3 (1 : 200) were used as secondary antibody staining reagents for IF staining, while a goat polyclonal secondary antibody recognizing mouse IgG (1 : 500, Abcam, ab150113) was used for immunohistochemistry; DAPI-containing TBST was applied for 5 min. Sections were visualized using an Olympus microscope (Olympus BX53, Olympus Corporation).

Cells were grown on glass coverslips and fixed with methanol and acetone (4:1) for 10 min at −20 °C. After washing 3 times with PBS supplemented with CaCl₂ (0.7 mM) and MgCl₂ (1.1 mM), cells were blocked with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature (RT), and incubated with anti-DOG1 primary antibody (1:200) in 1% BSA overnight at 4 °C. Binding of the primary antibody was visualized by incubation with a secondary antibody conjugated with Alexa 488 (1:500) in 1% BSA for 1 h at RT (Life Technologies, A-21206). Nuclei were stained with Hoechst 33342 (0.1 μg/mL PBS, Aplichem, Darmstadt, Germany). Cells were mounted on glass slides with mounting medium (DAKO Cytomation, Hamburg, Germany) and examined with an Axiovert 200 microscope equipped with ApoTome and AxioVision (Zeiss, Germany). Cellular distribution of endogenous TMEM16A was analyzed in CFBE or Cal33 cells in the presence or absence of CK2α’. Membrane and cytosolic expression were quantified in each cell by analyzing fluorescence intensities in the regions of interest (ROI) using the software ImageJ. Membrane regions were validated using high-resolution DIC image, that allowed us to clearly identify the plasma membrane of each cell.

For immunohistochemistry experiments, cryostat sections were rehydrated twice with 1X PBS containing 0.2% Triton (PBS-T). Antigen retrieval was performed using sodium citrate (pH 6) heated at 80 °C for 15 min. Sections were washed twice in PBS-T before being blocked in PBS-T containing 2% BSA. Next, sections were incubated with the following primary antibodies: rabbit anti zebrafish L-plastin (kindly provided by Dr Michael Redd^{74 (link)}) and/or mouse anti-PCNA (1:100; clone PC10, Dako; RRID: M0879) overnight at room temperature. The slides were then washed twice in PBS-T and incubated with DAPI (4′,6′-diamidino-2-phenylindole) and secondary antibodies: donkey anti-rabbit Alexa Fluor 488 for L-plastin (1:300; REF: A21206; Life Technologies, Bethesda, MD; RRID: AB_10049650) and goat anti-mouse Alexa Fluor 594 or 488 for PCNA (1:300; REF: A11005 for Alexa 594 and A28175 for Alexa 488; Life Technologies; RRID: AB_141372 and AB_2536161) for 1h30 at room temperature. Sections were rinsed and the slides were mounted with anti-fading medium (IMM Ibidi; REF: 50001). Note that PCNA antibody allows the detection of proliferative cells along the neurogenic niches as previously described^{75 (link)}, and L-Plastin antibody the detection of microglial cells, displaying ramified processes when quiescent, or amoeboid morphology when activated (phagocytic functions)^{76 (link)}.

For immunofluorescence staining, antigen retrieval of deparaffinized tissue sections was performed by microwave on the middle for 20 min. After being blocked with 5% donkey serum (Solarbio, Beijing, China) for 1 h at room temperature, the sections were incubated with primary antibodies at 4°C overnight, followed by incubation with anti-rabbit Alexa Fluor 488 (A21206; Life Technologies, Carlsbad, USA) or anti-goat Alexa Fluor 594 (A11058; Life Technologies) conjugated secondary antibodies. DAPI (Life Technologies) staining was performed for nuclear counterstaining. Fluorescent images were visualized by using a laser-scanning confocal microscope system (OLYMPUS, Tokyo, Japan). The following primary antibodies were used: goat polyclonal anti
-MCP-1 antibody (sc-1785; Santa Cruz Biotechnology, Dallas, USA) and rabbit polyclonal anti-Firefly Luciferase antibody (ab21176; Abcam, Cambridge, UK).