Immunofluorescence (AQP-4 Staining)

Sprague–Dawley rats were sacrificed 48 h after BBBD. Immunofluorescence was performed as described previously (Choi et al., 2019). Briefly, the brains of the rats were transcardially perfused with 0.9% sodium chloride (NaCl) followed by post-fixation overnight in ice-cold 4% formaldehyde. The fixed brains were dehydrated using 10%, 20%, and 30% sucrose gradients for cryoprotection. The frozen brain tissue was cut into 50-μm thick slices using a cryostat (CM1860, Leica, Nussloch, Germany), and tissue slices were permeabilized for 30 min in 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, United States). The permeabilized tissues were incubated for 2 h in a blocking solution containing 10% normal goat serum (Abcam, Cambridge, MA, United States), followed by overnight incubation with a rabbit monoclonal anti AQP-4 (Cat No. ab9512 1:250; Abcam, Cambridge, MA, United States). Subsequently, the slices were incubated with Alexa Fluor 488-conjugated mouse anti-rat IgG (Abcam, Cambridge, MA, United States) for 1 h at room temperature. The slides were mounted using a fluorescence mounting medium (Dako, Glostrup, Denmark). The tissue slides were scanned using a slide scanner (Pannoramic Scan II, 3D Histech, Budapest, Hungary), and the acquired images were processed using CaseViewer software (2.1v, 3D Histech, Budapest, Hungary). To quantify the AQP-4 values, we performed manual co-registration of AQP-4 and MR images to have comparable structures using MATLAB and analyzed AQP-4 values in the same ROIs as those in the MR image analysis.

Furthermore, we performed immunohistochemistry for the glial fibrillary acidic protein (GFAP) and AQP-4. The slides were rescanned by Axio Scan.Z1 microscope (Carl Zeiss, Göttingen, Germany) for the acquisition of high-resolution images. A magnified astrocyte was co-labeled with GFAP (red), AQP-4 (green), and DAPI (blue). The acquired images were post-processed using the Zen 2 image-processing software (blue edition, Carl Zeiss) and adjusted the values for brightness (white), gamma, and contrast (black) to reduce background noise. We applied the same parameters for all slides in both sonication and contralateral hemisphere.