PFA-fixed NSC cells were washed three times with PBS for 5 min. Then, they were incubated with 0.3% Triton X-100 for 10 min and blocked with 5% bovine serum albumin for 1 h at room temperature. Afterwards, cells were incubated with primary antibodies at 4 °C overnight. The primary antibodies were used as follows: anti-GFAP antibody 1:100 (AB5804; Millipore Corporation, Danvers, MA, USA), anti-nestin (1:100, ab81462, Abcam, Cambridge, MA, USA), anti-MAP2 (1:200, MAB3418), and anti-SOX-2 (1:200 sc-365823, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After rinsing the samples with PBS, the samples were then incubated with corresponding secondary antibodies for 1 h at room temperature. Nuclei were stained using 4,6-diamidino-2-phenylindole (DAPI, 1:1000; Life Technologies, Mulgrave, VIC, Australia).
Sc 365823
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-365823 is a laboratory equipment product from Santa Cruz Biotechnology. It is designed for use in scientific research and experiments. The core function of this product is to [insert short, factual, and unbiased description of the product's core function without extrapolation].
Automatically generated - may contain errors
Lab products found in correlation
Sc 5279, by Santa Cruz Biotechnology (2 mentions)
Sc 53108, by Santa Cruz Biotechnology (2 mentions)
Bs 12276r, by Bioss Antibodies (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Ab81462, by Abcam (1 mentions)
Ab5804, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Sc 166933, by Santa Cruz Biotechnology (1 mentions)
αpou5f1, by Santa Cruz Biotechnology (1 mentions)
T8787, by Merck Group (1 mentions)
Sc 8426, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti ki67 antibody sc 23900, by Santa Cruz Biotechnology (1 mentions)
Sc 10804, by Santa Cruz Biotechnology (1 mentions)
Sc 59987, by Santa Cruz Biotechnology (1 mentions)
Sc 18887, by Santa Cruz Biotechnology (1 mentions)
Sc 2025, by Santa Cruz Biotechnology (1 mentions)
Ab80892, by Abcam (1 mentions)
Anti rabbit or anti mouse secondary antibody, by Proteintech (1 mentions)
12 protocols using sc 365823
1
Immunocytochemical Characterization of NSC Cells
2
Immunofluorescence Analysis of Pluripotent Stem Cells
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12,000 cells were seeded in wells of 48-well plates coated with 0.1% gelatin and cultured in ESCs standard medium. After 72 h, cells were fixed with 4% paraformaldehyde for 15 min in darkness at RT and washed two times with PBS (Dulbecco’s Phosphate Buffered Saline, D8537; Sigma-Aldrich). Then, cells were permeabilizated with 0.25% Triton X-100 (T8787; Sigma-Aldrich) diluted in PBS for 5 min at RT followed by two times of PBS washes and blocking with 10% BSA (BP9702-100; Thermo Fisher Scientific) diluted in PBS for 30 min at 37°C. The antibodies αPOU5F1 (1:1,500, sc-5279; Santa Cruz Biotechnology), αSSEA1-488 (1:1,000, MA1-022-D488; Thermo Fisher Scientific), αZFP281 (1:1,000, sc-166933; Santa Cruz Biotechnology), αNANOG (1:500, sc-374103; Santa Cruz Biotechnology), and αSOX2 (1:1,000, sc-365823; Santa Cruz Biotechnology) were diluted in 3% BSA and incubated overnight in darkness at 4°C. To detect non-labelled primary antibodies, a rhodamine red-x secondary antibody was used (715-295-151; Jackson ImmunoResearch) for 1 h in darkness at RT. Nuclei were stained with DAPI (4′,6-diamidino-2-phenilindole, D9542; Sigma-Aldrich).
3
Immunofluorescence Analysis of hCECs
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The hCECs were cultured in a slide chamber and treated with ROCK inhibitors and incubated for 24 h. After removing the media, they were washed with phosphate-buffered saline (PBS), and fixed for 20 min in 3.7% (v/v) formaldehyde solution. Cells were permeabilized for 10 min with 0.5% (v/v) Triton X-100 and blocked for 1 h with 1% (w/v) bovine serum albumin (BSA) at room temperature. After washing with PBS, the cells were incubated overnight with either rabbit anti-Ki67 antibody (sc-23900, Santa Cruz, CA, USA), ZO-1 (sc-10804), β-catenin (ab32572), integrin β1 (sc-18887), SOX2 (sc-365823), N-cadherin (sc-59987), or E-cadherin (sc-8426) at 4 °C, and then washed with PBS. Cells are incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibody (1:100) or anti-mouse IgG antibody for 1 h at 37 °C in the dark. Cells were counterstained with Hoechst33342 nuclear staining dye. After extensive washing with PBS, the slides were mounted in a drop of mounting medium to reduce photobleaching. Negative control staining was conducted in parallel with the omission of the primary antibodies.
4
Chromatin Immunoprecipitation of Sox2 in ESCs
5
Immunofluorescence Assay for Pluripotency Markers
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For immunofluorescence assays, cells were fixed with 4% paraformaldehyde for 15–30 min at room temperature. The fixed cells were washed twice with PBS, incubated with PBS containing 0.1% Triton X-100 for 10 min, and washed three times with PBS. After blocking in BSA-blotting buffer (1% BSA and 0.1% Tween 20 in PBS) for 30 min, cells were incubated in BSA-blotting buffer with primary antibodies, including anti-Oct4 (1:200, sc-5279, Santa Cruz), anti-Sox2 (1:200, sc-365823, Santa Cruz), anti-Nanog (1:200, ab80892, Abcam), anti-SSEA-1 (1:200, 4744 S, Cell Signaling Technology), anti-Fibronectin (1:100, 15613-1-AP, Proteintech) and anti-Collagen-IV (1:50, 19797-1-AP, Proteintech), in a humidified chamber at 4 °C overnight. After washing three times, cells were stained for 1 h with either anti-mouse or anti-rabbit secondary antibody (1:200, Proteintech, China). Nuclei were stained with Hoechst33342 (10 μg/mL) for 2–5 min. Microscopy was performed on a Leica fluorescence microscope.
6
Protein Expression Analysis of A549 Cells
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Collected A549 and A549/GR cells were lysed in RIPA buffer (150 mM NaCl, 1% Nonidet p-40, 50 mM Tris, pH 8.0, and a protease inhibitor cocktail). Thirty micrograms of each sample was separated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, USA). Western blotting was performed with rabbit or mouse antibodies against Prx II: LF-PA0091 (AbFrontier, Seoul, South Korea), CD133: PA2049 (Boster Bio, CA, USA), Nanog: sc-293121, OCT3/4: sc-9081, Sox2: sc-365823 (Santa Cruz Biotechnology), VEGFR2: sc-6251, E-cad: sc-7870, Vimentin: sc-6260 (Santa Cruz Biotechnology), Shh: 2207 s, Gli-1: 3538 s, Notch 1: 3268 s (Cell Signaling Technology), CXCR4: YF-MA16239, STAT3: LF-MA30485,pSTAT3 (Tyr 705): LF-PA20474, pSTAT3 (Tyr-727): LF-PA20473 Hes-1: YF-MA11051, and β-Catenin: YF-MA10213 (AbFrontier). Protein expression levels were detected using Super signal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, #34577).
7
Immunohistochemical Analysis of Glioma Biomarkers
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IHC staining of tissue sections was performed as previously described (Man et al., 2018 (link)). Tissue microarrays including normal brain and low-grade and high-grade gliomas were purchased from US Biomax. The staining intensity of MBD3 was scored using a modified histochemical scoring (H-score) system to assess both the intensity of the staining and the percentage of positively stained cells. Briefly, for the intensity, a score of 0 to 3 (corresponding to negative, weak, moderate, or strong staining) was recorded, and the percentage of positively stained cells at each intensity was estimated. The H-score was calculated as [1 × (weak %)] + [2 × (moderate %)] + [3 × (strongly stained %)]. Images were acquired with a 20×/0.75 objective on a Hamamatsu 2.0 HT digital slide scanner; the acquisition software was Nanozoomer 2.0 HT. The following antibodies were used: MBD3 (1:50, 14258-1-AP, Protein Tech group, RRID:AB_2139745), STAT1 (1:500, 14994, Cell Signaling, RRID:AB_2737027), IFN-α (1:500, ab193055, Abcam), IFN-β (1:1,000, ab84258, Abcam, RRID:AB_1859645), p21 (1:100, 2947, Cell Signaling, RRID:AB_823586), and SOX2 (1:100, sc-365823, Santa Cruz, RRID:AB_10842165).
8
Western Blot Analysis of Organoid Markers
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Western blot analysis was performed, as described elsewhere [8] . Briefly, at least 16 pooled organoids per each condition were washed three times with PBS (pH 7.4) and lysed in buffer containing 50 mM Tris-HCl (pH 6.8), 10% glycerol and 1% sodium dodecyl sulfate (SDS). The lysates were homogenized by sonication and protein concentrations were determined using DC Protein Assay (Bio-Rad). The lysates were then supplemented with 0.01% bromophenol blue and 1% β-mercaptoethanol and denatured at 100 °C for 5 minutes. The prepared samples were separated by SDSpolyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore). The PVDF membrane was blocked in 5% skimmed milk in Tris-buffered saline containing Tween for 1 hour and incubated with primary antibodies overnight at 4 °C. The following antibodies were used: PAX6 (sc-53108, Santa Cruz, 1:1000), SOX2 (sc-365823, Santa Cruz, 1:1000), SOX1 (bs-12276R, Bioss, 1:1000), β-ACTIN (#4970S, Cell Signaling Technology, 1:1000).
9
Immunohistochemical Staining of Tissue Sections
10
Immunofluorescence Analysis of Cryopreserved Blastocysts
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Immunofluorescence analysis was performed as reported by Lee et al. (21 (link)). Re-expanded or hatched blastocysts in 24-h culture after freeze–thawing were fixed with 4% (v/v) paraformaldehyde, washed with DPBS, and permeabilized with 0.5% (v/v) Triton X-100 for 30 min. The blastocysts were then co-incubated with a blocking solution and SOX2 primary antibody (sc-365823, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, United States) overnight at 4°C. After rinsing with washing medium (Tween20, Triton X-100, and DPBS), the expanded blastocysts were incubated with the secondary antibodies: goat anti-mouse IgG (H + L) Alexa FluorTM 488 (A11029, 1:200; Invitrogen Corporation, Carlsbad, CA, United States), donkey anti-rabbit IgG (H + L) Alexa FluorTM 594 (A21207, 1:400; Invitrogen) for 1 h at 20°C–25°C. The nuclei were stained with Hoechst-33342 and examined under an epifluorescence microscope using the ZEN 3.5 Blue Edition software (Zeiss, Germany).
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