Anti-alpha smooth muscle Actin antibody (ab5694)

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Publication protocol

Corneas were blocked for 2 hours at room temperature in 3% BSA + 1.5% Triton-X, and then incubated with SMA (1:100) for two days at 4°C. After 2 days, corneas were rinsed 5 × 1 hour (for a total of 5 hours) in PBS at room temperature and incubated overnight with Topro3 (Thermo Fisher scientific; Waltham, MA, USA) and FITC-conjugated donkey anti-rabbit IgG (1:100). Finally, the cornea was flattened on a slide by applying 4 cuts in the peripheral cornea and mounting with mounting media (Vectashield: Vector Labs; Burlingame, CA). Negative controls, where the primary antibody was omitted, were run with all experiments. At least 3 corneas per condition were observed. Images were captured on a confocal microscope (TCS-SP5: Leica Microsystems; Bannockburn, IL).

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