Refine kit

Immunostaining of sponge slices was performed using a Leica Staining Robot with robotic antigen retrieval for F4/80 using trypsin solution (0.5 mg/mL in PBS, 10 min, 37 °C), or for αSMA manual antibody retrieval using a pressure cooker under standard conditions (citrate NCL pH 6 buffer); antigen retrieval was not performed for CD31. For F4/80 and αSMA, samples were blocked (5 min) with hydrogen peroxide solution from the Leica Refine Detection Kit (Leica Biosystems, Milton Keynes, UK) followed by serum block (30 min; F4/80, Immpress anti-rat (mouse absorbed) kit (Vector Laboratories, Peterborough, UK); αSMA, Mouse on Mouse Abcam Kit (Cambridge, UK)). Thereafter, samples were incubated with primary antibody (30 min; F4/80 1:300 (eBiosciences, Hatfield, UK); αSMA 1:4000 (Sigma-Aldrich, Dorset, UK)) prior to incubation with polymer (30 min, F4/80 Impress Kit as above; αSMA as above). For CD31 a Leica Refine Kit (as above) was used, which included a hydrogen peroxide block (5 min), primary antibody (120 min, 1:200 (Abcam)) and polymer incubation (15 min). Immunostaining was completed with incubation with 3,3′-diaminobenzidine (10 min) and counterstaining with haematoxylin (5 min), both from the Leica Refine Kit.