2.3. Immunofluorescence staining, confocal microscopy and colocalization analysis

To generate high-resolution images of the AQP8 protein and identify its subcellular localizations, a V5 tag was fused to the C-terminus of the AQP8 cDNA and overexpressed in RINm5F cells using lentiviral transduction. Cells were seeded on glass cover slips overnight, fixed with 2% PFA for 20 min and blocked with 1% BSA in PBS/0.1% TritonX for 1 h. To visualize AQP8, the V5 tag was stained by the primary V5 antibody (1:400) (E10/V4RR, Invitrogen, Carlsbad, CA, USA) in 0.1% BSA in PBS/0.1% TritonX using a Cy3-labelled secondary antibody (1:200) (Dianova, Hamburg, Germany) in 0.1% BSA in PBS/0.1% TritonX. To determine colocalization, the cells were simultaneously antibody-stained either with Con A (Concanavalin A) conjugated with Alexa Flour 488 ({"type":"entrez-nucleotide","attrs":{"text":"C11252","term_id":"1536323","term_text":"C11252"}}C11252, Thermo Fisher Scientific) for 1 h at RT after fixation and before permeabilization for the plasma membrane. The cells that were stained with Con A were also pre-incubated with either 100 μM H₂O₂ or with 100 μM Bt₂cAMP (N⁶,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt, D0627, Sigma-Aldrich, St. Louis, MO, USA) for 15 min before fixation. All other immunostainings were performed after the permeabilization and blocking procedure with either the COX IV (cytochrome c oxidase subunit 4) antibody (1:1,000) (#4844; Cell Signaling Technology) for the mitochondria, or with ABCD3 (1:200) ((F1):sc-514728, Santa Cruz Biotechnology, Dallas, Tx, USA) for the peroxisomes, with ERO1LB (1:100) (11261-2-AP, Thermo Fisher Scientific) for the ER (Endoplasmic reticulum), Giantin (1:1000) (PRB-114C, Covance, Princeton, NJ, USA) for the Golgi apparatus, or with Insulin (1:100) (ab7842, abcam, CA, UK). As secondary antibodies for the organelle specific staining, Alexa Flour 488 (1:200) (Dianova) were used. After antibody staining, the cells were stained with 300 nmol DAPI (4′,6-diamidino-2-phenylindole) in PBS for 5 min and embedded in ProLong Glass Antifade Mountant (Thermo Fisher Scientific). Imaging was performed using the Leica SP8 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) equipped with the Leica HCX PL APO 63x/1.40-0.60 oil objective (Leica Microsystems). Excitation was done via diode lasers at 552 nm for the visualization of Cy3, 488 nm for Alexa Flour 488, and 405 nm for DAPI. We used the Leica Application Suite X (LASX) software, version 3.5.7.23225 (Leica Microsystems) to adjust and capture the images also using z-stack imaging of 0.2 μm distances and 8-11 slices. To quantify colocalization, we used JACoP (just another Colocalization Plugin) [32] from Fiji to quantify the Pearson's correlation coefficient of 3D images.