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===Day 3=== #Wash with 1X PBS once #Add 200ul cold BS(PEG)9 (12ul stock BS(PEG)9 + 588ul 1X PBS prepared on ice) to sample and incubate 1hr at RT #Wash with 1X PBS twice #Add 200ul 1M Tris (pH 8.0) for 30min at RT #Wash with 1X PBS twice ====Imaging==== #Add 66ul of 0.5uM FISSEQ_Adpt Cy3 in 2X SSC + 30% formamide pre-heated to 75C #Incubate for 10min at RT #Wash with 2X SSC twice #*Lost 2 embryos, only the largest embryo (#18) left #Mount onto glass slide with 120um spacer between coverslip #*Use microscope mounting medium *552 Laser 1% *Cy3 Gain: 550 *Tilescan 4x5 tiles with 10% overlap *To image whole embryo with max resolution (2k x 2k with 0.3um step size) takes ~5hr
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