The deposition of endogenous ECM proteins onto uncoated and ECM-coated TCPS plates by H9 hESCs and 19-9-11 iPSCs was assessed via immunocytochemical staining. Samples were fixed in 10% formalin (Sigma) and permeabilized using 1% Triton X-100 (Sigma) in PBS. Primary antibodies against human fibronectin (Santa Cruz Biotechnology, catalog no. SC80559), laminin (Abcam, catalog no. ab30320), α-5 laminin (Millipore, catalog no. MAB1924), vitronectin (Santa Cruz Biotechnology, catalog no. SC74485), collagen type IV (Santa Cruz Biotechnology, catalog no. SC52317), and collagen type 1 (Sigma, catalog no. C2456) were diluted 1:500 in 3% BSA in PBS and allowed to incubate overnight at 4°C, followed by rinsing in PBS and incubation with Alexa Fluor 488- and 568-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Life Technologies, catalog nos. A10667 and A11031, respectively) in 3% BSA at room temperature for 1 hr. Cell nuclei were counterstained with 1 μg/ml DAPI (4′,6-diamidino-2-phenylindole dihydrochloride, Life Technologies, catalog no. D1306), and fluorescent images were captured using a Nikon IX51 epifluorescence microscope.
Vitronectin
Manufactured by Santa Cruz Biotechnology
Vitronectin is a glycoprotein that functions as a cell adhesion and spreading factor. It plays a role in various biological processes, including cell-matrix interactions, tissue remodeling, and coagulation. Vitronectin is commonly used in cell culture systems to promote cell attachment and growth.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Collagen type 1, by Merck Group (1 mentions)
Collagen type 4, by Santa Cruz Biotechnology (1 mentions)
Formalin, by Merck Group (1 mentions)
Laminin, by Abcam (1 mentions)
Human fibronectin, by Santa Cruz Biotechnology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using vitronectin
1
ECM Deposition on hESC and iPSC Cultures
2
Protein Quantification and Western Blot Analysis
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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 |
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