Goat serum

Cells were cultured on 8-well glass chamber slides (Millicell® EZ SLIDE 8-well glass, sterile, Catalog No. PEZGS0816, Millipore, USA) in the same manner as described for the experiments performed in tissue culture plates. Cells were pre-treated with 9 mM MSO for one hour prior to the addition of 1 μg/kg LPS. Cells treated with LPS alone served as positive controls, whereas cells treated with MSO alone or left untreated served as negative controls. After 6 h of LPS stimulation, the medium was aspirated, and cells were washed three times with PBS before being fixed to the chamber slides with a 4% paraformaldehyde solution in PBS for 10 min. Cells were made permeable by the addition of a 0.1% Triton X-100 solution in PBS for 20 min, followed by an overnight incubation with a monoclonal antibody specific for mouse IL-6 (Cell signaling technology, USA, Catalog No. D5W4V) at 4 degrees. Cells were washed before incubation with an Alexa Fluor-conjugated secondary antibody (Abcam, USA, Catalog No. ab15007) in the presence of 10% goat serum (R&D systems, USA). A mounting medium containing the nuclear stain DAPI (Electron Microscopy Sciences, USA, Catalog No. 17985–50) was applied to a cover slip, and fluorescence was visualized under a confocal microscope.

The cell-containing hydrogels were fixed in 4% (v/v) paraformaldehyde (Sigma-Aldrich, Castle Hill, NSW, Australia) for 45 min. Blocking and permeabilization were achieved by incubation with 5% goat serum (Gibco, ThermoFisher Scientific, Scoresby, VIC, Australia) and 0.1% Triton-X100 (Merck Millipore, Bayswater, VIC, Australia) in phosphate-buffered saline (PBS) for 2 h at room temperature. Primary antibody staining against the endothelial marker CD31 (cat No. bba7, R&D Systems; 1:200 in 1% goat serum) was performed overnight at 4°C. Subsequently, the samples were washed in 1% goat serum in PBS for 8 h with 3 changes of the washing buffer. Polyclonal goat anti-mouse IgG conjugated to Alexa-Fluor 488 (cat No. A11001, Invitrogen, ThermoFisher Scientific, Scoresby, VIC, Australia; 1:300) secondary antibody, Alexa-Fluor 633 conjugated Phalloidin (Invitrogen, ThermoFisher Scientific, Scoresby, VIC, Australia; 1:100), and 5 μg mL⁻¹ 4′, 6-diamidino-2-phenylindole (DAPI) in 1% goat serum/PBS were applied overnight at 4°C. Images were acquired on a Nikon A1R inverted confocal microscope (Nikon Instruments Inc.; 10x, 1.32 × 1.32 μm px⁻¹, z-step size 2.5 μm × 181). Image analysis was performed on the AlexaFlour-488 (green) channel of the acquired images to analyze the networks formed by the microvascular endothelial cells.

YS sections were baked onto slides for 2 hours at 60°C before
being dewaxed in xylene and rehydrated through graded ethanol as previously
described (10 (link)). Slides were washed with
distilled water then placed in a pressure cooker with boiling citrate buffer pH
6 (10 mM citric acid (Sigma), 0.05% v/v Tween 20 (Sigma) in DI water) for 2 min
for antigen retrieval. Slides were then washed for 3 min with distilled water
followed by 3 min in PBS (Sigma). Sections were blocked with 20% (v/v) goat
serum (R&D Systems) for 45 min at room temperature. Primary antibodies
were diluted in blocking solution (data S23), added to the sections and incubated for 1 hour
at room temperature. Slides were washed twice for 3 min each in a wash buffer
(0.1% (w/v) Triton X (Sigma) in PBS), then twice for 3 min each in PBS.
Secondary antibodies (see data
S23) were diluted in blocking solution, added to section and
incubated for 2 hours at room temperature. The wash step was repeated and then
300 nM DAPI (Sigma) was added. Slides were incubated for 5 min before washing
with PBS. Slides were then mounted with ProLong™ Diamond Antifade
(Thermofisher) and imaged on a Zeiss Axioimager with Zeiss ZEN pro software.