To validate the close proximity (<40 nm) between two proteins in gastric cancer cells, PLA was achieved by using Duo-link In Situ-Fluorescence kits according to the manufacturer’s instructions (Sigma-Aldrich). The MKN45 cells were grown on 8 chamber slides (Corning, New York, 354108), fixed in 4% paraformaldehyde, and permeabilized using 0.1% Triton-X-100. Cells were then incubated with blocking solution for 1h and incubated overnight with primary antibodies at 4°C (anti-DARPP-32 (sc-398144), Santa Cruz) plus anti-IGF1R (IGF1R (9750s), Cell Signaling,). The cells were subsequently incubated with PLA PLUS and MINUS probes for mouse and rabbit and incubated with ligation-ligase solution for 60 min at 37 °C, and subsequently with amplification-polymerase solution according to the manufacturer’s instructions. By mounting solution with DAPI, the slides were mounted. Each red spot represents the close proximity of two interacting proteins. Cell images were obtained using an Inverted laser-scanning LSM880-Airyscan (Zeiss, Thornwood, NY) confocal microscope.
8 chamber slides
Manufactured by Corning
Sourced in United States
The 8 chamber slides are a laboratory equipment product designed for cell culture and various biological applications. The slides feature eight separate compartments, allowing for the simultaneous cultivation and observation of multiple samples. The chambers are constructed with high-quality materials to provide a controlled and consistent environment for cell growth and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Duo link in situ fluorescence kits, by Merck Group (2 mentions)
Anti darpp 32 sc 398144, by Santa Cruz Biotechnology (2 mentions)
Igf1r 9750s, by Cell Signaling Technology (2 mentions)
Lsm 880 airyscan, by Zeiss (2 mentions)
Alexa fluor 488 conjugated anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bz x700 all in one fluorescence microscope, by Keyence (1 mentions)
Alexa fluor 594 conjugate, by Thermo Fisher Scientific (1 mentions)
Dmi6000 microscope, by Leica (1 mentions)
Human fc block, by BD (1 mentions)
4 protocols using 8 chamber slides
1
Validating Protein Proximity in Gastric Cancer
2
Immunofluorescence Imaging of Cellular Stress Response
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Approximately 1 × 104 cells were seeded in 8-chamber Slides (Corning) 24 h after siRNA or plasmid transfection. Cells were treated with ABS (pH 4, 200 μM) for 20 min and recovered for designated time points. After the treatment, cells were fixed with fresh-made 4% formaldehyde for 45 min. Then immunofluorescence staining was performed as previously described [35 (link)]. Antibodies for IGFBP2 (Cell signaling), EGFR (Invitrogen) and Phospho-DNA-PKcs (Thr2609) (Invitrogen) were incubated at 4 °C for overnight. Alexa fluor-568 conjugated anti-rabbit and Alexa fluor-488 conjugated anti-mouse secondary antibody (Invitrogen) were incubated for 1 h at room temperature. Cells were mounted with DAPI solution. Images were captured using the All-in-one Fluorescence Microscope (BZ-X700) (Keyence, Itasca, IL, USA).
3
Validating Protein Proximity in Gastric Cancer
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To validate the close proximity (<40 nm) between two proteins in gastric cancer cells, PLA was achieved by using Duo-link In Situ-Fluorescence kits according to the manufacturer’s instructions (Sigma-Aldrich). The MKN45 cells were grown on 8 chamber slides (Corning, New York, 354108), fixed in 4% paraformaldehyde, and permeabilized using 0.1% Triton-X-100. Cells were then incubated with blocking solution for 1h and incubated overnight with primary antibodies at 4°C (anti-DARPP-32 (sc-398144), Santa Cruz) plus anti-IGF1R (IGF1R (9750s), Cell Signaling,). The cells were subsequently incubated with PLA PLUS and MINUS probes for mouse and rabbit and incubated with ligation-ligase solution for 60 min at 37 °C, and subsequently with amplification-polymerase solution according to the manufacturer’s instructions. By mounting solution with DAPI, the slides were mounted. Each red spot represents the close proximity of two interacting proteins. Cell images were obtained using an Inverted laser-scanning LSM880-Airyscan (Zeiss, Thornwood, NY) confocal microscope.
4
Assessing Endothelial Cell-Monocyte Interactions
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HNDMVECs were seeded on 8-chamber slides (354118, Corning, NY) at 5000 cells/cm2 and cultured 7 days to form a confluent monolayer. The sheared or statically incubated G-THP-1 cells were then added to a monolayer of HNDMVECs (1×106 cells per well), followed by incubation for 6 hours. The cell cocultures were washed twice with sterile PBS, fixed with formalin. For staining, the fixed cells were permeabilized with 0.1% Triton-X-100 for 10 minutes, blocked with 1% BSA and human BD Fc block (BD Bioscience, Franklin Lakes, NJ) for 1 hour, followed by incubation with 1:200 vascular endothelial cadherin (VE-cadherin) monoclonal antibody (16B1), Biotin (Thermofisher) for 1 hour, 1:500 streptavidin, Alexa Fluor 594 Conjugate (Thermofisher) for 45 minutes. Nuclei were stained with DAPI. The slides were then washed, mounted, and imaged using a Leica DMI6000 microscope with Leica SP8X confocal. VE-cadherin staining was quantified using ImageJ [28 (link)].
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