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[DESCRIPTION]
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<p>This is an In-Cell Western™ Assay protocol for the detection of Phospho-p53 in COS cells in response to Hydroxyurea.</p>
<p> </p>
<p><strong>Developed for:</strong></p>
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<p>Aerius,<br /> Odyssey® Classic,</p>
<p>Odyssey CLx, and</p>
<p>Odyssey Sa<br /> Infrared Imaging Systems</p>
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<p><strong>Please refer to your manual to confirm that this protocol is appropriate for the applications compatible with your Odyssey Imager model.</strong></p>
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[GUIDELINES]
<p><strong>Required Reagents</strong></p>
<p><strong>LI-COR Reagents</strong></p>
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<p>IRDye® 800CW Goat anti-Rabbit Secondary Antibody (LI-COR P/N 925-32211 or 926-32211)</p>
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<p>IRDye 680RD Goat anti-Mouse Secondary Antibody (LI-COR P/N 925-68070 or 926-68070)</p>
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<p>Odyssey® Blocking Buffer (LI-COR, P/Ns 927-40000 or 927-50000)</p>
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<p><strong>Additional Reagents</strong></p>
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<p>1X PBS wash buffer</p>
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<p>Tissue culture reagents (serum, DMEM, trypsin, 1X PBS)</p>
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<p>Clear or black 96-well or 384-well microplates (See Experimental Considerations)</p>
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<p>Hydroxyurea (Sigma®, P/N H8672)</p>
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<p>Anti-phospho-p53 (Cell Signaling Technology, P/N 9286)</p>
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<p>Normalization antibody: Anti-total ERK1 (Santa Cruz Biotechnology, P/N SC-94)</p>
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<p>20% Tween® 20</p>
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<p>37% formaldehyde</p>
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<p>10% Triton® X-100</p>
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<p><strong> Experimental Considerations</strong></p>
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<p>Proper selection of microplates can significantly affect the results, as each plate has its own characteristics, including well depth, plate autofluorescence, and well-to-well signal crossover. Use the following general considerations for microplate selection.</p>
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<p>In-Cell Western analyses use detection at the well surface with minimal liquid present. This results in minimal well-to-well signal spread, allowing the use of both clear as well as black-sided plates with clear bottoms. Do not use plates with white walls, since autofluorescence from the white surface will create significant noise.</p>
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<p>In-Cell Western assays require sterile plates for tissue culture growth. The following plates are recommended by LI-COR Biosciences:</p>
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<p>                 96-well format, Eppendorf (P/Ns 0030730119 or 0030741030)</p>
<p>                 96-well, clear, Nunc® (P/Ns 167008, 161093)<br />                 96-well, clear, FalconTM (P/Ns 353075, 353948)<br />                 96-well, black with clear bottom, Nunc (P/N 165305)</p>
<p>                 96-well format, Corning (P/N 3603)</p>
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<p>                384-well, clear, Nunc (P/Ns 164688, 164730)</p>
<p>                384-well, clear, Falcon (P/Ns 353229, 353289)</p>
<p>                384-well, black with clear bottom, Nunc (P/N 142761)</p>
<p>                384-well format, Corning (P/N 3712)</p>
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<li><strong>Focus Offset Optimization</strong> – If plates other than those recommended are used, the focus offset can be determined empirically by scanning a plate containing experimental and control samples using the following focus offset settings.</li>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/yxdesfe.png" width="593" height="137" data-src="https://s3.amazonaws.com/pr-journal/yxdesfe.png" data-ofn="Screen Shot 2018-05-31 at 11.44.24 AM.png" /> </p>
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<p>Use the same intensity settings for each scan. After reviewing the scans, use the focus offset with the highest signal-to-noise ratio for experiments. The actual minimum and maximum focus offset will vary with each instrument. Alternatively, the plate manufacturer can be consulted to obtain the measured distance from the skirt bottom to the bottom of the plate.</p>
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<li>All Aerius and Odyssey® Imaging systems (excluding Odyssey Fc) require microplates that have a maximum 4.0 mm distance from the base of the microplate to the target detection area of the plate (actual maximum focus offset varies with each Aerius and Odyssey Sa instrument and is found by choosing Settings &gt; System Administration, then clicking Scanner Information). When using plates specified for In-Cell Western assays, the recommended focus offset is 3.5 mm or higher.</li>
<li><strong>Intensity Setting Optimization -</strong></li>
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<p> <img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/yxfesfe.png" width="606" height="242" data-src="https://s3.amazonaws.com/pr-journal/yxeesfe.png" data-ofn="Screen Shot 2018-05-31 at 11.45.18 AM.png" /></p>
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<p>Protect plates from light before imaging to ensure highest sensitivity. When storing plates after imaging, the plates should remain protected from light at 4 °C.</p>
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<p><strong> Experimental Results</strong></p>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g9vc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/g9vc2w6.png" data-ofn="Screen Shot 2016-12-14 at 11.29.37 AM.png" /></p>
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