Immunofluorescence staining was performed as previously described (Gu et al., 2014 (link)). Cell samples were fixed with 4% (w/v) paraformaldehyde (PFA; P6148, Sigma) for 10 min and then permeabilized with 0.5% Triton X-100 (Sigma) and blocked in 2% BSA (Sigma) at 37°C for 10 min after rinsing. Samples were subsequently incubated with primary antibodies against OCT4 (Santa Cruz Biotechnology, sc-8628, 1:200), SOX2 (Santa Cruz, sc-17320, 1:200), TRA-1-60 (Millipore, MAB4360, 1:200), TRA-1-81 (Millipore, MAB4381A4, 1:200), SSEA4 (Millipore, MC-813-70, 1:200), BEST1 (Abcam, ab14929, 1:150), TH (Santa Cruz, sc-14007, 1:200), PAX6 (Abcam, ab5790, 1:200), OTX2 (Millipore, AB9566, 1:200), MESP1 (Aviva System Biology, OAAB09387, 1:50), CTNT (Abcam, ab8295, 1 μg/mL), TUJ1 (Cell signaling, 2125), LMX1α (Abcam, ab31006, 1:50), SOX17 (R&D Systems, AF1924, 1:200), or HNF4α (Epitomics, 2803-1, 1:200) at 4°C overnight. On day 2, the samples were rinsed and then incubated with the appropriate secondary antibodies donkey-anti-mouse-cy5 (Jackson ImmunoResearch, 103856, 1:200), donkey-anti-goat-Cy5 (Millipore, AP1805A6, 1:200), donkey-anti-rabbit-FITC (Jackson ImmunoResearch, 99320, 1:200), or donkey-mouse-cy3 (Jackson ImmunoResearch, 715-165-150, 1:200) for 1 hr at 37°C. Nuclei were visualized by staining with Hoechst 33342 (10 μg/mL) or propidium iodide (5 μg/mL) for 10 min at room temperature.
Sc 17320
Manufactured by Merck Group
Sc-17320 is a laboratory equipment product. It is designed to perform specific functions in a research or analytical setting. The core function of this product is to facilitate various scientific experiments and analyses, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (1 mentions)
Donkey anti mouse cy5, by Jackson ImmunoResearch (1 mentions)
Donkey anti rabbit fitc, by Jackson ImmunoResearch (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
P6148, by Merck Group (1 mentions)
Mab4360, by Merck Group (1 mentions)
Sc 8628, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab9260, by Merck Group (1 mentions)
Ab1550, by Merck Group (1 mentions)
Ab144p, by Santa Cruz Biotechnology (1 mentions)
2 protocols using sc 17320
1
Immunofluorescence Staining Protocol for Pluripotency and Lineage Markers
2
Immunohistochemical analysis of brain and eye
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Animals were transcardially perfused with ice-cold PBS (pH 7.4) followed by 4% paraformaldehyde in PBS (pH 7.4) at different postnatal stages, then brains and eyeballs were dissected out and post fixed in 4% paraformaldehyde overnight at 4 °C. After washing in PBS for 1 h, the samples were immersed in 30% sucrose in PBS until saturation, followed by cryosection at 40μm thickness. The sections were washed by PBS for 3 times and blocked with blocking buffer (10% DS, 0.1% Triton, 1 × PBS) at room temperature for 2 hours. The sections were then incubated with the primary antibody at 4 °C overnight. The antibodies used for immunostaining were activated-Caspase3 (CST, 9664 s, 1:500), AXL (Santa Cruz, SC-1096, 1:200), Calretinin (Millipore, AB1550, 1:200), ChAT (Millipore, AB144P, 1:200), Sox2 (Santa Cruze, sc-17320, 1:200), Ki67 (Millipore, AB9260, 1:200), GFAP (CST,3670 s, 1:500) and Z631 (link) (2 μg/ml). After rinsing with 0.1%PBST, secondary antibody was incubated for 2 hours at room temperature. Finally, after sections were washed with 0.1%PBST, nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) (Invitrogen,1:500).
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