TITLE: Spatial Transfer of Oligonucleotides and Imaging
AUTHORS: Sanjay Srivatsan, Mary  Regier 

[STEPS]
?. [Prep work]
Prepare the following reagents prior to beginning the protocolNuclei Buffer (NB)Indexed Cold Lysis Buffer (CLB)5% PFA solution Nuclei Buffer + SuperaseIn +  BSA (NSB)
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A single mouse embryo section (stored at -80C) and a single sci-Space grid (stored at -20C) were removed. The back of each was wiped to remove moisture. Outline on the back of each slide (side that does not contain the oligo grid or section) with a marks surrounding the section or the grid. ---------------------------------------------------------------------------------------------------------------------This is to help place the grid directly over the embryo.
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Indexed Cold Lysis Buffer Solution (CLB):Prepare   of cold lysis buffer solution for every slide used.If you are preparing solution for more than one slide prepare the pool in a 15mL conical.1uM DAPI (final concentration) 0.25% IGEPAL v/v   -- Make a 10% solution as a working stockSolvent: Nuclei Buffer ---------------------------------------------------------------------------------------------------------------------Mix well after preparation, keep cold and out of light as much as possible.
500 µl
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Nuclei Buffer (NB):10mM Tris/HCl pH 7.4 10mM NaCl 3mM MgCl2  Solvent: Nuclease Free water Prepare enough NSB for  mL per section and an additional 4mL to account for overage and washing nuclei after pooling
0.5 mL
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Wide-bore p1000 pipette tips. Cut the tip of p1000s or
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5% PFA solution: Make enough solution for 1mL per section.1.25x PBS d 5% PFA Solvent: Nuclease Free water ---------------------------------------------------------------------------------------------------------------------Mix well after preparation. The remaining PFA can aliquoted and stored at -20C for future use.Make sure to handle concentrated PFA solution in a fume hood.
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Place the embryo section on two transfer clips with the transfer clips surrounding the section.  The clips have a directionality -- place the rounded holders closer to yourself and the shaved tips away from you.  Note: The transfer clips are not crucial, but they are very helpful in securing the stacked slides during transfer.
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Using a wide-bore tip draw up 500uL of the indexed CLB solution and start by gently pipetting onto the embryo. As the embryo becomes covered in solution, move the pipette to cover the bottom of the slide. It's important not to introduce any bubbles during this stage.
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For each slide that you are planning to index, aliquot 495uL of the Cold Lysis Buffer into a separate 1.5mL tube.To each tube add 5uL of a 10uM slide-specific indexing oligo---------------------------------------------------------------------------------------------------------------------Note: Make sure that the slide oligo used is unique from any sequence found in the sci-Space grid.
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Use the sci-Space grid and align the marks made during step2. Carefully snap the grid onto the tissue section by lowering the bottom half of the sci-Space grid onto the section and then slowly pushing until the two slides are completely flush. At this stage it is also important that bubbles do not form. Press the stacked transfer with the thumbs and apply gentle pressure to evacuate any excess fluid between the two slide. Use of a napkin or KimWipe at this stage greatly aids in the removal of excess fluid.
?. [Imaging]
Place the stacked transfer onto the microscope for imaging. We used a Zeiss Axio Observer microscope with a 2.5x fluorescent objective.  At this stage using the microscope, the boundaries of the image were marked by the microscope and tiled images were captured on two fluorescent channels (DAPI and FITC).
?. [Cell recovery and fixation]
After imaging insert a razor blade between the two slides in the stack and lift to separate them. Place the used sci-Space grid aside and add 100uL of 5% PFA solution to the embryo. Allow the solution to sit for 15seconds before using a cell scraper  to scrape the solution into a 15mL conical tube. Use an additional 900uL of  the 5% PFA solution to recover all the material on the slide. Mix the entire 1mL solution with a few strokes of pipetting.---------------------------------------------------------------------------------------------------------------------The same cell scraper can be used for multiple slides provided that it is cleaned with some ethanol.
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Sonicate the solution for 10seconds using the bioruptor sonicator and then place on ice for 15 minutes.
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Prepare all slides in this manner using a different indexed CLB solution for each slide. We normally prepare slide 5 or 6 at a time. This means that the slides prepared first are sitting in fixation solution for the longest. We have not seen a negative impact on data quality as a result of this. Make sure to let the last slide prepared sit on ice for at least 15 minutes.
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Using a swinging bucket rotor in a chilled centrifuge, spin cells down at 800g for 10 minutes.
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Carefully remove all supernatant from all tubes and resuspend each pellets in 250 uL of NSB (Nuclei Buffer + SuperaseIN + BSA). Combine the pellets and centrifuge again at 800g for 6 minutes.
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Resuspend pellet in 1mL of NSB and flash freeze. Nuclei can be stored at -80C for at least one year.
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Nuclei Buffer + SuperaseIN + BSA (NSB) solution:1% SuperaseIN 1% v/v BSA  Solvent: Nuclei Buffer
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At the moment the sci-Space protocol has been optimized for OCT-embedded fresh-frozen sections. In the sci-Space study these sections were purchased from zyagen -- https://zyagen.com
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Video of this process --