Ac 40

Cells were lysed in radioimmunoprecipitation (RIPA) buffer (Thermo Scientific) and protein content was measured by bicinchoninic acid assay. Protein was separated by electrophoresis in 4-12% Bis-Tris polyacrylamide gels (NuPage, Life Technologies) and transferred to polyvinyl difluoride membranes. Membranes were probed for acetylated α-tubulin (6-11B-1, 1:1000; Abcam) and actin (AC-40, 1:2000; Abcam). The bound primary antibodies were detected by chemiluminescence with HRP-conjugated secondary antibodies (1:10,000; Millipore).

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.

Hela and Sf9 cells were cultured on 13 mm washed coverslips and then fixed using 4% fresh paraformaldehyde in PEM buffer (8 mM PIPES pH 6.8, 5 mM EGTA, 2 mM MgCl₂). Alternatively, Hela cells were fixed in 100% methanol (from −20 °C stocks) for 10 minutes at 4 °C, followed by 10 minutes at room temperature. After fixation, cells were stored in PBS at 4 °C. Paraformaldehyde fixed cells were permeabilised using 0.1% Triton-X 100, and blocked with 1% BSA in PBS. Cells were labelled with biotinylated Affimers, diluted in PBS containing 1% BSA to a final concentration of 2.5 μg/ml, and incubated for 1 hour. The coverslips were then washed, fluorescently labelled streptavidin (ThermoFisher) at a dilution of 1/100 was added in PBS, and the cells incubated for 1 hour. Coverslips were washed in PBS, and coverslips mounted on glass slides in Prolong Antifade (ThermoFisher). Paraformaldehyde fixed cells were additionally co-stained for F-actin using fluorescent phalloidin (Molecular probes), or for non-muscle myosin 2B (Sigma).
For methanol fixed cells, a mouse monoclonal anti-actin antibody (AC-40, Abcam) was added at 1/100 dilution together with the diluted Affimer in the first incubation, and an Alexa dye-labelled anti-mouse secondary antibody (Thermofisher) was added in the second incubation at a dilution of 1/400.