Immunohistochemistry imaging

Scaffold were washed 3 times in PBS and fixed in a 3.7% (v/v) solution of formalin and PBS for 10 min, followed by 3 additional washes. Permeabilization was performed using a 0.05% TWEEN in a 10 mM Tris and 1 mM MEDTA solution, scaffolds were incubated in 300 µl for 1 h before washing 3 times.

Scaffolds were dehydrated in 2-propanol solutions graduating from 30 to 100% for 10 min in each. Scaffolds were then left in a solution of 2-propanol and polyester wax (1:1) at 50 °C overnight. Next, scaffolds were placed in polyester wax for 3 h and then fresh wax overnight at 50 °C. Scaffold were halved and blocked for sectioning into 35 µm slices.

Cell were stained for DNA using 0.1 mg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, UK) in PBS for 10 min. IHC was performed to establish the presence of key cell types including, primary antibodies aquaporin-1, aquaporin-2 and synaptopodin (Stratech, UK) were used at a 1 µg/ml dilution, von Willebrand factor (Abcam, UK) was used at 2 µg/ml (Fig. 1), and scaffold were incubated overnight in 10 µl, no-primary controls were used. Alexa Fluor 488 anti-rabbit IgG (ThermoFisher, UK) was used as a secondary antibody and left to incubate for 1 h before performing 3 washes, 5 min each. Imaging was done using a Zeiss Axio Imager fluorescence microscope.

A diagram of the kidney showing the structure of kidney including the nephron and glomerulus, highlighting the location of key cell types, representative IHC images are taken from large scaffold at 7 days, scale bar is 100 µm