Isolectin b4

Cryosections (7 µm in thickness) were stained with TRITC-conjugated wheat germ agglutinin (WGA, Sigma-Aldrich, Taufkirchen, Germany) to outline cardiomyocytes and with Isolectin B4 (Vector Laboratories, Newark, CA, USA) to visualize endothelial cells and capillaries. Fibrosis was detected with the Sirius Red staining method.
Immunofluorescence staining was performed using standard procedures. Antibodies used for immunofluorescence staining were: hosphor-p38 MAPK (#9211), hosphor-Akt #(9271) (both from Cell Signaling Technology, Danvers, MA, USA), α-actinin (A7811, Sigma-Aldrich, Taufkirchen, Germany) followed by Anti-Rabbit/Mouse IgG Alexa Fluor^® 488 (#4412), or Anti-Rabbit/Mouse IgG Alexa Fluor^® 555 secondary antibody (#4409, NEB, Ipswich, MA, USA). Nuclear staining was performed with Mounting Medium (#HP20.1, Roth, Karlsruhe, Germany) with DAPI.

For analysis of capillary and arteriole density, gastrocnemius muscle sections (7 μm) were stained with isolectin-B4 (Vector Laboratories, B-1205; 1:100) to identify endothelial cells and the smooth muscle marker, α-smooth muscle actin conjugated to Cy3 (Sigma-Aldrich, C6198, 1:100) as previously described^{19 (link)}. High resolution images were acquired (at × 200) and counts from 18 randomly selected fields were averaged and expressed as the number of capillaries and arterioles (< 50 μm) per mm² of muscular sections^{20 (link)}.
DNA fragmentation, associated with apoptosis, was detected in sections (7 μm) of gastrocnemius muscle using the commercially available terminal deoxynucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) kit (Click-iT Plus TUNEL Alexa Fluor 594 kit, Life Technologies). Sections were counterstained with Isolectin B4 to identify endothelial cells and DAPI to label nuclei.
Skeletal tissue fibrosis was detected in transverse sections (7 µm) of gastrocnemius muscle using Mason’s Trichrome staining.

Explant tissues were cultured, washed, and stained using standard protocols as mentioned above. For the pericyte coverage analysis, the endothelium in the metatarsal assay was stained with an anti-CD31 (BD Pharmingen, 553370) antibody and in the aortic ring assay with Isolectin B4 (Vector Labs, FL-1201). Pericytes were stained with anti-αSMA (Sigma, C6198) and anti-NG2 (Millipore, AB5320) antibodies. Single images were taken using a 20 × objective focussed on the plane of vessels using a Nikon epifluorescent microscope. For quantitative analysis, using NIS Elements Software, single thresholds were calculated for each individual channel (endothelium, pericytes) and the intersection of the endothelium covered by pericytes. The analysis was blinded to treatments and was performed on masked images with different thresholds used per image as required to avoid saturation. Pericyte coverage was then calculated as % (Intersection/Endothelium), and the values were normalised to the average of the internal control (vehicle/PBS) for every experiment. The normalised values of at least three independent experiments were pooled for statistical analysis. Outliers were eliminated using the average ± 2SD mean.

The retinal vasculature was visualized by immunohistochemistry on retinal whole mounts in line with previous works 22. Briefly, the retinal whole mounts were freeze‐thawed and incubated overnight at 4°C in fluorescein labelled isolectin B4 (1:200; Vector Laboratories, Burlingmae, CA, USA). Retinal whole mounts were examined for changes in their vascular pattern with a microscope equipped with epifluorescence (Eclipse Ni‐E; Nikon, Amsterdam, The Netherlands). Images of the retinal vasculature were acquired through a digital photocamera (DS‐Fi1c camera; Nikon) and processed using an image‐editing software (Adobe Photoshop; Adobe Systems, Inc., Mountain View, CA, USA) to create whole retina montages in which the extent of the avascular area and the total area of pre‐retinal neovascular tufts were measured according to previous quantification studies 34. For each experimental condition, quantitative data originated from six retinas from six different mice either WT or AQP4 KO.

Animals were deeply anaesthetized by intraperitoneal injection of pentobarbital (150 mg/kg bodyweight) and eyes enucleated using curved forceps. For retinal flatmount preparation, eyes were prefixed with 4% PFA for 20 min at 4 °C following excision of the cornea, lens, sclera and vitreous and isolation of the retina. Retinas were flatmounted on permeable cell culture inserts (BD Falcon) and sequentially fixed with 4% PFA from both sides for 20 min at 4 °C, respectively. For immunohistochemical staining retinal flatmounts were washed with 0.1 M phosphate buffer (PB) and then incubated with a blocking and permeabilization solution for 30 min at RT shaking. Tissue was incubated with primary antibodies against CD31 (rat, 1:100, BD Biosciences) and Isolectin B4 (1:100, Vector Labs), overnight at 4 °C. The next day the tissue was washed and incubated with corresponding secondary antibodies conjugated to Cy3 (Thermo Fischer) for 2 h at RT. All antibodies were diluted in blocking and permeabilization solution (TNB, 0.1% TBST, 3% normal goat serum). Flatmounts were mounted in 0.1 M PB on Superfrost Plus^TM microscope slides (Thermo Fisher) and coverslipped using Mowiol.