Fixing and decalcifying Pocillopora and Porites tissue for LCM, to further improve sectioning and extractions

Protocol here

1. Day 1 (7/3/24): Fixed tissue in PAXgene fixative (Pocillopora and Porites compressa (frag Test3) from CBLS Wetlab)
   1. Relax colony in 3 % w/volume magnesium chloride
      1. To make solution, filter seawater from CBLS wetlab through a 0.2 um filter. Transfer to clean and labelled screwtop glass bottle.
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      2. Add 3 g MgCl2 to each 100 mL of seawater
      3. Warm bottle to tank temperature for ~30 mins.
      4. Fill a 200 mL beakers with solution and transfer one fragment per beaker to try to relax the tissue
         1. this honestly does not look like it is working as we intend. We want the polyps to stick out but they contract after a few minutes in sthe solution anyways…
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   2. Then cut off branches and transfer gently to PAXgene fixative for 24 hours in fume hood, room temp
      1. Porites: cut off 3 branches and put into fresh PAXgene tissue container (10 mL removed for POC below)
      2. Pocillopora: put 2 branches each in a 5 mL tube with 5 mL PAXgene fixative each
2. Day 2 (7/4/24): Replace fixative with stabilizer, transfer to 4 ºC
3. Day 3 (7/5/24): Started decalcification, kept on shaker in cold room (4 ºC)
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4. Day 5 (7/7/24): Changed EDTA solution
5. Day 6 (7/8/24), POC samples: POC Decalcification done, tissue cryoprotected
   1. Transfer into 10 mL of 15% RNAse-free sucrose at 4 ºC in RNAse-free PBS until the tissue sinks
      1. Sank immediately
   2. Then into 30 % sucrose in RNAse-free PBS at 4 ºC overnight (did not have dry ice to embed so left in sucrose until 7/10/24)
6. Day 6 (7/8/24), POR samples: Changed EDTA solution
7. Day 7 (7/9/24), POR samples: Changed EDTA solution
8. Day 8 (7/10/24), POR samples: POR Decalcification done, tissue cryoprotected
   1. This was an almost 120 hour (5-day) decalcification, which is the longest I’ve ever gone
   2. Transfer into 10 mL of 15% RNAse-free sucrose at 4 ºC in RNAse-free PBS until the tissue sinks
      1. Sank immediately
   3. Then into 30 % sucrose in RNAse-free PBS at 4 ºC for 4 hours, until sink
9. Day 8 (7/10/24): Embed POC and POR on powdered dry ice and store at -80 ºC until sectioning
   1. Tightly wrapped in aluminum foil cleaned with Ethanol and RNAse away, then aluminum package wrapped tightly in ziplock and stored at - 80 ºC in a plastic box.
10. Sectioning and LCM Notes: https://zdellaert.github.io/ZD_Putnam_Lab_Notebook/LCM-Test-4/