Protein was isolated from cell culture and human tissue (retina, RPE, and choroid) and total protein was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA), as previously described55 (link). The death to preservation time for the human donor tissue was <8 h. Western blotting was performed as previously described with the housekeeping protein, beta-actin (Table 2 ), serving as a loading control58 (link).
Antibodies/stains used for western blot, immunofluorescence, and histology.
| Antibody/stain | Source | Dilution | Catalog# |
|---|---|---|---|
| Alpha-Smooth muscle actin (αSMA) | Sigma-Aldrich | 1:5000 | A5228 |
| Apolipoprotein E (APOE) | Millipore | 1:500 | AB947 |
| Beta-Actin | SCBT | 1:5000 | sc-47778 |
| CD31 | Dako | 1:50 | M0823 |
| Complement factor H (CFH) | Quidel | 1:200 | A312 |
| Hoechst 33258 pentahydrate | Invitrogen | 2 μg/ml | H3569 |
| Iba1 | Wako | 1:200 | 019-19741 |
| Osteopontin (OPN) | Sigma-Aldrich | 1:200 | O7264 |
| Vitronectin (VTN) | Santa-Cruz | 1:500 | SC-74484 |
| Von-Kossa method for calcium kit | Polysciences | 24633-1 |
Free full text: Click here
