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<p><strong>Developed for:</strong></p>
<p> </p>
<p>Aerius,<br /> Odyssey® Classic,</p>
<p>Odyssey CLx, and</p>
<p>Odyssey Sa</p>
<p>Imaging Systems</p>
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[GUIDELINES]
<p><strong>Required Reagents</strong></p>
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<p><strong>LI-COR Reagents</strong></p>
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<p>IRDye® 800CW goat anti-mouse secondary antibodies (LI-COR P/N 925-32210 or 926-32210)</p>
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<p>IRDye 680RD goat anti-rabbit secondary antibodies (LI-COR P/N 925-68071 or 926-68071)</p>
</li>
<li>
<p>Odyssey® Blocking Buffer (LI-COR P/N 927-40000 or 927-50000)</p>
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<p> </p>
<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>Poly-D-Lysine coated 96-well microplate clear/black or clear (Corning P/N 354640</p>
<p>or 354461)</p>
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<li>
<p>Heparin (CALBIOCHEM®, P/N 375097)</p>
</li>
<li>
<p>Acidic Fibroblast Growth Factor (Upstate Group Inc., P/N 01-116)</p>
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<p>Primary antibodies</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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<div class="column"><em>SPECIAL NOTE: NIH3T3 cells do not adhere strongly to TC-treated plates, resulting in the need for poly-D-lysine-coated plates in this assay. However, even with lysine-coated plates, the adherence of cells remains relatively weak compared to other cell lines.</em></div>
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<div class="column"><em>Be extremely cautious and delicate in handling plates and pipetting when washing, removing, and adding solutions to avoid detaching the cells.</em></div>
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<div class="column"><strong>Experimental Considerations</strong></div>
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<p>Establish the specificity of the primary antibody by screening plate-like lysates through Western blotting and detection on an Odyssey instrument. If significant non-specific binding is present, choose alternative primary antibodies to avoid results with non-specific signal detection.</p>
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<p>Proper selection of microplates can significantly affect 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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<li>
<p>In-Cell Western analyses use detection at the well surface with no liquid present. This results in minimal well-to-well signal spread, allowing the use of both clear and black-sided plates with clear bottoms. <strong>Do not use plates with white walls, since autofluorescence from the white surface will create significant noise.</strong></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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<ul>
<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.<img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/yxjesfe.png" width="596" height="143" data-src="https://s3.amazonaws.com/pr-journal/yxjesfe.png" data-ofn="Screen Shot 2018-05-31 at 12.40.57 PM.png" /></li>
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<p><br />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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<p> </p>
<p>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.</p>
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<p><strong>Intensity Setting Optimization -</strong></p>
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<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/yxkesfe.png" width="570" height="279" data-src="https://s3.amazonaws.com/pr-journal/yxkesfe.png" data-ofn="Screen Shot 2018-05-31 at 12.41.42 PM.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>
</li>
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<p><strong>IV. 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/g9wc2w6.png" width="450" data-src="https://s3.amazonaws.com/pr-journal/g9wc2w6.png" data-ofn="Screen Shot 2016-12-14 at 12.04.33 PM.png" /></p>
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