Anti-integrin immunofluorescence staining was performed on cryosections of hPLAP-prestained osteochondral explants as described previously.31 (link),32 (link) In brief, sections were fixed with ice-cold acetone for 2 min at −20°C and blocked with 5% normal goat serum (Vector Laboratories) in PBS supplemented with 0.2% Tween-20 for 30 min at RT. Cryosections were stained overnight at 4°C for collagen I (rabbit polyclonal; Rockland Immunochemicals), collagen II (rabbit polyclonal; Rockland Immunochemicals), α1 integrin (hamster clone Ha31/8; BD Biosciences), α2 integrin (mix of three rat clones; Emfret), and α5 integrin (rabbit polyclonal; Santa Cruz Biotechnology), or a combination of α2 integrin and collagen I or collagen II for co-immunofluorescence staining, followed by incubation with FITC-conjugated goat anti-rabbit (Sigma), Alexa 488-conjugated goat anti-rat (Molecular Probes), Texas Red-conjugated goat anti-hamster (Vector Laboratories), and Cy3-conjugated goat anti-rabbit (Jackson ImmunoResearch), either single or in mixture, for 1 h at RT. Stained sections were analyzed and pictures were taken with a microscope (Axioskop 2; Zeiss).
Integrin α2
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Integrin-α2 is a protein involved in cell adhesion and signaling. It functions as a receptor for collagen, laminin, and other extracellular matrix proteins.
Automatically generated - may contain errors
Lab products found in correlation
Integrin α5, by Santa Cruz Biotechnology (2 mentions)
Alexa 488 conjugated goat anti rat, by Thermo Fisher Scientific (1 mentions)
Axioskop 2, by Zeiss (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Fitc conjugated goat anti rabbit, by Merck Group (1 mentions)
Cy3 conjugated goat anti rabbit, by Jackson ImmunoResearch (1 mentions)
Collagen 1, by Rockland Immunochemicals (1 mentions)
α tubulin, by Merck Group (1 mentions)
Amersham ecl, by GE Healthcare (1 mentions)
Vimentin, by Merck Group (1 mentions)
E cadherin, by BD (1 mentions)
Immobilon psq, by Merck Group (1 mentions)
Cyclin d1, by Santa Cruz Biotechnology (1 mentions)
Anti cd44 apc, by BD (1 mentions)
Integrin β1, by Santa Cruz Biotechnology (1 mentions)
P fak, by Santa Cruz Biotechnology (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
Nanog pe, by BD (1 mentions)
Mrp1 fitc, by BD (1 mentions)
Aldh1 fitc, by BD (1 mentions)
6 protocols using integrin α2
1
Integrin-Mediated Osteochondral Extracellular Matrix Visualization
2
Western Blot Analysis of Cell Proteins
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Cultured cells, washed with cold PBS, were scratched with RIPA buffer (Tris 20 mM pH 8.8, NaCl 150 mM, EDTA 5 mM, Triton X-100 1%, and protease inhibitors) and supernatant boiled at 100°C for 5 min with Laemmli buffer. The same amount of protein was resolved by 10% SDS-PAGE and transferred onto a PVDF membrane (Immobilon PSQ, Millipore, Massachusetts, USA). Membranes were blocked in TBS with 5% nonfat dried milk and 0.1% Tween 20 for 1 h prior to incubation at 4°C overnight with the appropriate primary antibody (1 : 200 α2-integrin, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 400 cyclin D1, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 50 vimentin, Sigma-Aldrich, Saint Louis, Missouri, USA; 1 : 2000 E-cadherin, BD Transduction Laboratories, Palo Alto, CA, USA; 1 : 1000 α-tubulin, Sigma-Aldrich, Saint Louis, Missouri, USA). Detection was done using horseradish-peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagents (Amersham ECL, GE Healthcare, Buckinghamshire, UK).
3
Flow Cytometric Analysis of NSCLC Stem Cell Markers
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Expression of human NSCLC stem cell marker CD133 and CD44 was analyzed by flow cytometric study in A549 cells and spheroids by using anti-CD133-FITC and anti-CD44-APC antibodies (BD Biosciences). CD133+ and CD44+ CSCs were flow-cytometrically gated from spheroids on the basis of the cell surface phenotype. Mean fluorescence intensities of Oct-4-PerCP-Cy5.5, Nanog-PE, Sox-2- Alexa Fluor-647, Mrp1-FITC, Aldh1-FITC (BD Biosciences) were quantified22 (link). Mean fluorescence intensities of Akt, p-Akt, Cxcr-4, Mdr1, Integrin-α2, Integrin-α5, Integrin-β1, p-Fak (Santa Cruz Biotechnology, Inc.) were determined with respective primary antibodies conjugated with PE as previously described13 (link).
4
Osteosarcoma Protein Expression Analysis
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The total proteins of osteosarcoma cells were extracted by 1× SDS gel protein buffer (Solarbio, Beijing, China). Then, samples were cooked at 95°C to denature the proteins. The protein was transferred to PVDF membrane (Solarbio, Beijing, China) using an electrotransfer system according to standard procedures. After transferring the membrane, the PVDF membranes were blocked with 0.5% skimmed milk powder (diluted in PBS solution) for 1 hour at room temperature, and the primary antibody was incubated at 4°C overnight. The primary antibodies are list: integrin α2 (1:100, Santa Cruz, Texas, USA), integrin β1 (1:1000, CST, Massachusetts, USA), JAK1 (1:1000, Abcam, Cambridge, UK), p-JAK1 (1:1000, Abcam, Cambridge, UK), JAK2 (1:1000, Abcam, Cambridge, UK), p-JAK2 (1:1000, Abcam, Cambridge, UK), STAT3 (1:1000, Abcam, Cambridge, UK), p-STAT3 (1:1000, Abcam, Cambridge, UK) and actin (1:1000, Abcam, Cambridge, UK). After that, the HRP secondary antibody (1:5000, Abcam, Cambridge, UK) were incubated at room temperature for 1 hour, and finally treated with super-sensitive luminescent solution (Solarbio, Beijing, China).
5
Integrin and Connexin Protein Analysis
6
ELISA-based Integrin-Lumican Binding Assay
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Lumican was coated onto the wells of Maxisorp 96-well microtiter plates and incubated for 18 h at 4°C. Each well was washed three times with PBST (0.2% Tween-20 in PBS), then the plates were blocked with 1% BSA in PBST for 2 h. Cell lysates were added to the plates, incubated for 2 h, then the wells were washed three times (Kim et al., 2018 (link); Park et al., 2019 (link)). Preparations of integrin α2 (Santa Cruz Biotechnology) or β1 antibody (Cell Signaling Technology) in blocking solution were added to the plates and allowed to react for 2 h. After washing, HRP-linked antibody (Cell signaling Technology) was added and the lysates were incubated for 2 h, and then washed five times. The reaction was developed with 100 μl TMB substrate solution and stopped with 100 μl of 1 N H2SO4. Microtiter plates were measured at 450 nm using a microplate reader (Infinite 200 PRO, Tecan Life Sciences, Zurich, Switzerland).
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