Immunofluorescence studies for the detection of AGR2 (1 in 100 dilution) (Abcam, Cat No: ab227584), WFDC2 (1 in 250 dilution) (Abcam, Cat No: ab200828) and TSC22D3 (1 in 50 dilution) (Abcam, Cat No: ab197987) were performed as described previously [24 (link),33 (link),34 (link)]. Epithelial expression of AGR2, WFDC2 and TSC22D3 positive cells was confirmed using cytokeratin (1 in 500) (Biocare, Denmark) and appropriate Alexa fluorophor conjugated secondary antibodies (Thermo Fisher Scientific, Austin, TX, USA). Localization of AGR2 protein expression to the endoplasmic reticulum (ER) was confirmed using the ER marker GRP94 (1 in 1000 dilution) (Abcam, Cat No: ab210960).
Ab210960
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab210960 is a recombinant monoclonal antibody produced in Chinese Hamster Ovary (CHO) cells. The antibody is directed against human Toll-like receptor 4.
Automatically generated - may contain errors
Lab products found in correlation
Ab227584, by Abcam (1 mentions)
Ab200828, by Abcam (1 mentions)
A0063, by Agilent Technologies (1 mentions)
Axio imager 2, by Zeiss (1 mentions)
Anti c3d a0063, by Agilent Technologies (1 mentions)
Ab109201, by Abcam (1 mentions)
Ab68418, by Abcam (1 mentions)
Ab125011, by Abcam (1 mentions)
Enhanced chemiluminescence solution, by Beyotime (1 mentions)
Ab92324, by Abcam (1 mentions)
Bicinchoninic acid bca assay kit, by Beyotime (1 mentions)
Ab52649, by Abcam (1 mentions)
Ab16502, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
Ab86299, by Abcam (1 mentions)
4 protocols using ab210960
1
Immunofluorescence Analysis of AGR2, WFDC2, and TSC22D3
2
Immunofluorescence Staining of Cellular Markers
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Cells seeded on coverslips were fixed with a 3% formaldehyde solution for 15 min at RT, washed and quenched with 50 mM NH4Cl for 10 min before being permeabilized with PBS-0.1% TRITON X-100 for 10 min at RT and blocked for 1 h with PBS-1% BSA FcR Blocking Reagent (130-059-901, Miltenyi). Cells were incubated with primary antibody at 4 °C overnight: anti-C3d (A0063, DAKO), anti-GRP94 (ab210960, Abcam) or anti-GM130 (AF8199, R&D System), washed and stained with secondary fluorescent antibodies. Images were acquired as described above and analyzed by Zen 2.3 Lite® software.
3
Protein-Protein Interaction Analysis by PLA
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PLA™ was performed with Duolink® In Situ Orange Starter Kit Mouse/Rabbit (DUO92102-1KT) following manufacturer’s protocol. The primary antibodies couples used were: anti-C3d (A0063, DAKO) and anti-GRP94 (ab210960, Abcam, Cambridge, UK), anti-C3d (A0063) and anti-HSP105 (sc-74550, SantaCruz Biotechnology, Dallas, Texas, USA), anti-GRP94 (ab210960) and anti-TLR4 (56580, NOVUS Biological, Centennial, Colorado, USA) and anti-cathepsin-L (PA5-88413, ThermoFisher Scientific, Waltham, Massachusetts, USA). Microscopy images were taken on an Axio Imager 2 (Carl Zeiss Microscopy GmbH, Jena, Germany). Images were acquired using an AxioCam MRm monochrome CCD camera (Carl Zeiss GmbH) and analyzed using ImageJ® software. Spotted quantification was performed using Icy® software.
4
Quantifying Extracellular Vesicle Proteins
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Total proteins were extracted with RIPA lysis buffer and then qualified with the Bicinchoninic Acid (BCA) assay kit (Beyotime, Beijing, China). Thereafter, protein extracts (20–40 μg) were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and then blocked with 5% milk for 1 h. Next, the membrane was probed with primary antibodies overnight at 4 °C, followed by the corresponding secondary antibody for 1 h at 37 °C. The blots were examined enhanced chemiluminescence solution (Beyotime). All antibodies were bought from Abcam (Cambridge, MA, USA): p65 (1:5000, ab16502), phosphorylated (p)-p65 (1: 5000, ab86299), TNFAIP3 (1:5000, ab92324), CD81 (1: 2000, ab109201), TSG101 (1:5000, ab125011), CD63 (1:2000, ab68418), GM130 (ab52649, 1:5000), GRP94 (1:1000, ab210960), and β-actin (1 : 5000, ab6276).
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