Antifade reagent containing dapi

Cell-based indirect immunofluorescence assay was performed as described previously with modifications (31 (link)). Human embryonic kidney (HEK) 293 cells (ATCC) were seeded onto 8-well chamber slides at 10,000 cells per well and cultured in DMEM in 5% CO₂ at 37°C. They were transfected with pAQP4 using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Cells were fixed with 4% paraformaldehyde, washed with PBS, and blocked with 1% BSA in PBS. Sera of mice were diluted at 1:10 with PBS containing 1% BSA. Commercially available rabbit anti–mouse AQP4 antibody (catalog A5971, Sigma-Aldrich) was diluted at 1:500 and used as positive control. To remove nonspecific antibodies, diluted sera were incubated with rat liver powder at room temperature for 1 hour and then centrifuged at 16,900g for 15 minutes. Next, 100 μL of the diluted sera were added to each well and incubated at 4°C overnight. After washing with PBS, cells were incubated with either Alexa Fluor 488–conjugated anti-mouse IgG (for samples) (catalog A21202) or anti-rabbit IgG (for positive control) (catalog A11070) secondary antibody (all from Thermo Fisher Scientific) at room temperature for 1 hour. Cells were coverslipped with antifade reagent containing DAPI (Thermo Fisher Scientific).

For sialic acid staining, 16HBE14o- or MDCK cells were seeded onto coverslips in 24-well culture plates at a density of 1×10⁵ cells/well and 1x10⁴ cells/well, respectively. Two days after seeding, the culture medium was removed and cells were washed twice with cold PBS and fixed with 4% paraformaldehyde for 20 min. Subsequently, cells were washed 3 times with PBS and incubated in PBS containing 10 μg/ml MAL I (Maackia amurensis lectin), for α-2,3 sialic acid receptor, or SNA (Sambucus nigra lectin), for α-2,6 sialic acid receptor, for 1 hr. Cells were then rinsed 3 times with PBS before the coverslips were mounted in antifade reagent containing DAPI (Thermo Fisher Scientific) to stain the nuclei. A negative control was obtained by incubating cells with PBS in the absence of primary antibody. Fluorescent imaging was performed by confocal laser-scanning microscope (FV1000; Olympus). Cells were excited at 358 and 495 nm and emission detected at 461 and 515 nm to evaluate DAPI and FITC fluorescence, respectively. To remove sialic acid receptors from the cell surface, cells were treated with 1 U/ml α-2,3 sialidase for 1 hr at 37°C prior to sialic acid staining.

The left lobes were inflated with 4% paraformaldehyde at 25 cm H₂O pressure. Paraffin-embedded tissue sections (5 μm) were then prepared and the sections were evenly divided into three groups. Sections were stained with hematoxylin and eosin (H&E) or stained with Alcian blue and PAS (AB-PAS).
For immunofluorescence staining, paraffin-embedded sections were dewaxed through xylene (2 changes) and rehydrated through descending alcohol (100%-95%-80%-70%) to deionized H₂O. Antigen recovery was performed by using pre-heated Na-Citrate buffer (10 mM, pH 6.0) for 10 min in the microwave. Sections were blocked in blocking buffer (3% BSA, 0.3% Triton X-100 in PBS) at room temperature for 1 h. Sections were incubated with primary antibody at 4 °C overnight, incubated with secondary antibody at room temperature for 1 h. Cover slips were mounted on stained sections with anti-fade reagent containing DAPI (Invitrogen, USA). The primary antibody used was Anti-Clara cell secretory protein (CCSP also known as CC10 OR CC16) (1:2000 dilution, 07-623, EMD Millipore), monoclonal anti-acetylated tubulin (1:10,000 dilution, T7451, Sigma), and monoclonal anti-α smooth muscle actin (1:400 dilution, A 2547, Sigma). The secondary antibody used was Alexa Fluor labeled goat anti-rabbit antibody, goat anti-mouse antibody (1:200 dilution, A-11008, A-11005, Thermo Fisher, USA).