Vectamounttm

Tissue sections were incubated at 60°C for 10 min to facilitate tissue adherence onto the slides before deparaffinization in two changes of xylene substitute (Sigma-Aldrich Co., St Louis, MO, USA) each for 15 min. This was followed by serial rehydration in graded ethanol (GmbH, Hamburg, Germany) from 100% ethanol followed by 70%, 50% and 30% ethanol, and finally in distilled water. Heat-mediated antigen retrieval was conducted in Tris-EDTA buffer (pH 9.0) using a microwave pressure cooker for 10 min followed by incubation with a mouse anti-TRPM4 monoclonal antibody (clone 10H5; Abcam, Cambridge, UK) at 1:500 dilution (1.536 μg/ml) for one hour at room temperature. Binding of the anti-TRPM4 antibody was detected using HRP-conjugated secondary anti-mouse/rabbit antibody from EnVision^TM detection system (DakoCytomation, Carpinteria, CA, USA) for 30 min and developed with DAB as the chromogen for 5 min. The sections were counterstained with fresh Gill No. 2 hematoxylin solution (Sigma Aldrich) for 10 sec and mounted with the VectaMount^TM (Vector Labs, Burlingame, California) non-aqueous mounting medium.

Manual RNAscope 2.5 HD Duplex assays were performed on formalin-fixed paraffin‑embedded sections (5-μm thickness) from P15 control (normoxic) and OIR mouse retinas according to the manufacturer’s protocols (ACDBio; Doc #322452-USM and #322500-USM). The following sets of RNAscope probes were used in this study: (i) Mm-Camk2g-C2 (Cat #522071-C2) together with Mm-Cldn5 (Cat #491611) and (ii) Mm-Camk2d-O1 (Cat #508941) together with Mm-Cldn5-C2 (Cat #491611-C2). Slides were lightly counterstained with Gill’s hematoxylin solution and mounted in VectaMountTM (Vector Laboratories Inc.). Images were acquired using a Nikon E400 microscope with ×40 or ×60 air objectives. Camk2g and Camk2d dot quantification (×60 magnification) in claudin-5 (Cldn5)-positive vascular endothelial cells, the retinal GCL, INL, and ONL was performed using ImageJ (65 (link)). Five images were obtained per slide from each mouse, where each slide contained 2–3 retinal sections. For each image, dots were quantified in 3 randomly selected regions of interest per retinal region. Data are summarized as the average number of dots per mm².