Accustain iron stain kit

The right lung was inflated in situ with 10 mL of 4% paraformaldehyde (PFA), removed, and embedded in paraffin for sectioning. To visualize iron deposits, lung sections (5 µm) were stained with Prussian blue using the Accustain® Iron Stain kit (Sigma-Aldrich).³⁹ Sections were examined by light microscopy and the number of iron particles was counted in a blinded fashion. In some sections, a double staining of von Willebrand Factor (vWF) and Prussian blue was conducted following antigen retrieval in sodium citrate buffer.^{40 (link)} Staining of endothelial cells was performed using a rabbit anti-vWF antibody (1:200 dilution) and developed following the manufactures’ instructions (Chemicon Blood Vessel Staining Kit ECM590). Once the desired color intensity was reached, the slides were subjected to the Prussian blue staining protocol described above.

Histology was done as described previously (Reuter et al., 2016 (link)). In short, animals were sacrificed within 3 days after MRI under deep Isoflurane anesthesia by transcardial perfusion with 4% acid free formalin (Roth, Karlsruhe, Germany). The harvested brains were incubated over night in 4% acid free formalin at 4°C, cut into blocs with 2 mm thickness, dehydrated with ethanol and xylol and embedded in paraffin. For histochemical analysis 4 μm sections were dewaxed in xylene and rehydrated in alcohol and distilled water.
For detection of cMBs Prussian blue (PB) staining was performed using the Accustain® Iron Stain Kit as described in the manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO, USA). Nuclei were counterstained using nuclear fast red 0.1% (Merck, Darmstadt, Germany) for 10 min. Following dehydration steps in alcohol and xylol the sections were preserved in mounting medium (Eukitt, O. Kindler, Freiburg, Germany).
Bright field analysis was done using a Leica DM 4500 B fluorescence microscope (Leica, Wetzlar, Germany). Pictures were taken with Leica IM50 Image Manager Software (Leica, Cambridge, UK).