Automated slide stainer

Immunohistochemistry was carried out as reported^{5 (link)}. Carotid arteries were harvested 7 days after arteriotomy from MSC-treated rats (n = 5) and from DMEM-treated rats (n = 5). Control carotids were harvested from uninjured rats (n = 5). Harvested vessels were fixed in 4% buffered formaldehyde, dehydrated and embedded in paraffin. Consecutive 4% formaldehyde-fixed 5 μm cross-sections were deparaffinised and rehydrated. Antigen retrieval was performed in a microwave through incubation in 10 mM citrate buffer pH 6.0. Endogenous peroxidases were blocked with 4% H₂O₂. Blocking was carried out in 5% donkey serum, followed by incubation with the primary HIF3α antibody (H-170), with the HIF1α antibody (H-206) (Santa Cruz Biotech.) or with CONFIRM anti-CD45RO (UCHL-1) primary antibody (Ventana Medical Systems Inc., USA) at 1:100 dilution at 4 °C overnight. Immunostaining was performed manually (HIF1α) or on a Ventana automated slide stainer in combination with the iView DAB detection kit and accessories according to manufacturer’s instructions. Primary antibodies were omitted in negative controls. Nuclei were counterstained with Mayer’s haematoxylin (Sigma-Aldrich). Image screening and photography were performed using a Leica microscope IM1000 System.