Histology/Immunohistochemical/Collagen/Elastin Staining

Fresh tissue (IVC with intact thrombus) was fixed in 10% buffered formalin for 2 hours and then transferred to 70% ethanol for subsequent paraffin embedding and slide mounting (5 μm thickness) as previously described.¹⁶ Antigen retrieval was performed using the heat-mediated sodium citrate method; 10 mM sodium citrate solution, pH 6.0 at 95°C for 10 minutes and then allowed to cool for 20 minutes. Nonspecific antigen-binding sites were blocked with normal serum, and sections were incubated with primary antibodies to Ly6G (551459, 0.5 ug/mL, BD Biosciences, San Jose, California, United States) and citrullinated histone3 (Cit-H3)(ab5103, 2 ug/mL, Abcam, Cambridge, Massachusetts, United States). A species-specific peroxidase kit (Vector Laboratories Inc., Burlingame, California, United States) was used according to the manufacturer’s instructions for the corresponding secondary antibody and 3,3′-diaminobenzidine (DAB) substrate application. The slides were counterstained with haematoxylin. In a blinded fashion, positive cells were counted and totaled in five high-power fields (hpf, 1,000 × ) radially around the IVC wall.

Picrosirius red staining to quantify collagen content was performed as previously described.¹⁶ These sections were then analysed in crossed-plane polarized light from a monochromatic source to assess cross-linked collagen. Six images for each were obtained using a Zeiss Axio Imager M1 microscope and Zeiss Zen 2 (blue edition) software (Carl Zeiss Microimaging GmbH, Göttingen, Germany) at 0 and 90° to the plane of polarization, to capture the birefringence of fibres extinguished in one direction. The images were analysed blindly utilizing the National Institutes of Health (NIH) Image J software. The area corresponding to the vein wall was selected as a region of interest, and then the image underwent threshold segmentation to differentiate collagen from other (mainly cellular and empty space) components of the vein wall. A vein wall collagen score was assigned by the formula [(%birefringent area) × (measured vein wall area)] / (total specimen area). Haematoxylin and eosin stain for vein wall thickness score, as previously reported,¹² and Verhoeff’s elastic stain for elastin visualization were performed in the University of Michigan Dental School Histology Core.