Ab108905

Blood obtained by cardiac puncture into 10% volume of sodium citrate (3.8%, w/v) was centrifuged for 15 minutes at 2300 g, and plasma was carefully collected from the supernatant fraction. Plasma samples were divided into aliquots, snap-frozen, and stored at −80°C until analyses. Plasma total antigen levels of tPA, PAI-1, and A2AP were measured by ELISA using kits according to the manufacturer’s instructions (catalog numbers are listed in Supplemental Methods). Plasma PAI-1–free tPA was measured by ELISA by first capturing the free tPA on a surface coated with PAI-1 antigen, enabling detection of only the functional, PAI-1–free form of tPA. tPA enzymatic activity in plasma or tissue lysates was assayed by chromatographically measuring the release of para-nitroaniline (pNA) chromophore from a plasmin-specific synthetic substrate (Abcam, ab108905). Results were recorded and analyzed by VersaMax Microplate Reader and SoftMax Pro software (Molecular Devices, Thermo Fisher Scientific).

Anti-Neuroserpin (Santacruz SC32947 and SC48360), anti-Plasmin (Santacruz 15036) and anti-methionine sulfoxide (Novus Biologicals NBPI06707SS) antibodies were used for probing western blots and immunohistochemistry of retinal sections. β-actin (Sigma, AC-40), anti-collagen (Abcam ab6586), anti-elastin (Abcam Ab23747) and anti-laminin (Abcam ab11575) antibodies were used for WB. Plasmin was from Sigma, Missouri, USA and recombinant neuroserpin protein from abcam. Plasmin enzyme activity was measured using 96-well microplate format kits from AnaSpec (Sensolyte Assay kit, AnaSpec, Inc., CA AS-72125). Fluorescent polystyrene microbeads were obtained from Invitrogen (FluoSpheres; Invitrogen, Carlsbad, CA) and dimethyl pimelimidate (DMP) was from Sigma, St. Louis, USA. Tissue type Plasminogen activator (tPA) (ab108905) and urokinase type Plasminogen activator (uPA) (ab108915) activity assay kits were obtained from Abcam. All other analytical grade reagents were from Sigma, USA.