Axio imager ax10

The part of aorta with maximum diameter were harvested on day 14 and embedded in Tissue‐Tek OCT compound (SAKURA 4583, Sakura Finetek, USA, Inc) The samples were frozen at −80°C. Then, 5‐um sections were fixed in pre‐cold 4% formaldehyde for 15‐20 minutes. The samples were blocked in 5% BSA in PBS for 1 hour. Next, Alexa 488 antimouse F4/80 (Biolegend 123119), antimouse NOS2 PE‐eFluor 610 (eBioscience) and Alexa Fluor 647 antimouse CD206 (MMR) (Biolegend 141711) antibodies were used to detect the expression of the M1 or M2 macrophage markers in the aortas. The incubations were performed at room temperature for 1.5 hour in a humidified box and protected from light. DAPI was used to stain the cell nuclei (blue) at a concentration of 1 µM. ProLong™ Gold Antifade Mountant (ThermoFisher, P36930) was applied to protect the fluorescence from fading. Images were taken using a Carl Zeiss Axio Imager AX10 with ZEN 2011 (blue edition). The fluorescence was measured using digital image analysis software (ImageJ v.1.41, National Institutes of Health). The percentage of iNOS⁺ and CD206⁺ cells relative to the F4/80⁺ cells was calculated and analysed in each group.

Formalin fixed paraffin embedded lung sections (4μm) were used for staining Elastica van Gieson (EvG) (sigma) was stained according to the manufacture’s protocol. Von Willebrand Factor (vWF) immunohistochemistry was performed as previously published [32 (link)]. Immunofluorescence studies of 5-LO (1:25) and LTA4H (1:50) were performed according to the protocol previously published [32 (link)]. Images were taken with AxioImager AX10, Axiocam MRm and Axiovision 3.1 software (Carl Zeiss, Göttingen, Germany) for the vWF and Elastica Van Gieson. Studies for immunofluorescence of 5-LO and LTA4H, optical sections were acquired by laser-scanning confocal microscopy with a Leica TCS-SP2 confocal microscope and images were arranged with ImageJ. The confocal microscopy was performed at the VCU Department of Anatomy and Neurobiology Microscopy Facility, supported, in part, by funding from a NIH-NINDS Center core grant (5P30NS047463-02).