Sectioning and performing LCM on the POC and POR branches that were prepared on 7/10/24, using the same steps outlined in this early June post

Modifying sectioning protocol to fix tissue to slides immediately after sectioning, following the PAXgene suggestions here

Modified Cresyl violet staining solution (from here)

1. Prepare Cresyl violet staining solution at least one week prior usage
2. Add 0.5 g Cresyl violet into 50 ml 100% ethanol
3. Mix solution and store at 4°C sealed air-tight and dark
   1. mixed on orbital shaker overnight in cold room, with occasional vigorous shaking to resuspend powder into solution

Sectioning: 7/17/24

1. Section onto PEN membrane slide for LCM as well as extra slides for confocal morpholgy imaging and backup for RNA and DNA extraction
2. All surfaces and equipment should be treated with RNAse cleaner!!!
3. Morning of: prepare PEN slide with UV and RNAse cleaner
   1. Make sure to not damage or touch the membrane in any way
   2. Using sterilized and RNAse zap-ped forceps, dip slides in RNAse zap for 15 seconds
   3. Follow this by two 15 second rinses in DEPC water
   4. Let dry, at room temperature or at 37 ºC
   5. When visibly dry, place in UV box for 30 minutes (ideally do so immediately prior to sectioning)
   6. With clean, gloved hands, transfer to slide box for sectioning
4. Prepare solutions:
   1. 70% Ethanol, 2 containers
   2. 100% Ethanol
5. Sectioning procedure:
   1. Keep slide cold in the cryostat, briefly warm sections with finger to adhere
   2. Air-dry the slide for 1 min at room temperature (15–25°C).
   3. 70% Ethanol, 1 minute (some protocols say 2 min)
   4. Transfer the slide to ice-cold 100% ethanol for transport to lab
6. In lab: Staining Procedure (modified/combined from here and here to reduce exposure to aqueous solutions of lower than 70% ethanol to reduce RNAses) (ALSO)
   1. ALL SOLUTIONS ICE COLD
      1. Place slide on petri dish
      2. Apply Cresyl violet staining solution (in 100% ethanol) directly with syringe and sterile filter to the section and incubate for 1 minute, swivel gently
      3. Dip for 5 seconds in 70% ethanol (dip several times to remove all OCT)
         1. Repeat with fresh container of 70% ethanol
         2. may need to dunk slide in RNAse-free H2O to remove all OCT. If so it needs to be very quick and ice cold and then immedately dunked in cold ethanol again.
      4. Dip for 5 seconds in 100% ethanol (dip a few times)
         1. Can fix up to 2 minutes in 100% ethanol or even for short-term storage prior to LCM
      5. Air dry sample 1-2 in drying chamber with desiccant or fume hood
         1. if possible, proceed to immediate LCM at this step!
7. Freeze slides at -80 ºC

This all went super well. I sectioned at -25ºC which worked really well for Porites, and less so for the pocillopora. But the pocillopora branch was also teeny and I had a lot of issues with it. But the POR worked great, and I got one PEN slide from each POC and POR and got two morphology slides from POC to view with the confocal. Adding a rinse step with ice cold RNAse free H2O during the stain washes was necessary but effective to remove OCT. Hopeful for LCM tomorrow.

LCM: 7/18/24

1. Buffers to collect cells in, bring to LCM and fill tube caps with 40 uL of solution when loading onto scope:
   1. Zymo Proteinase K digestion buffer (190 uL) + proteinase K (10 uL)
   2. Zymo RNA/DNA lysis buffer
   3. Charm DD1 frozen tissue buffer

This went well! Hard to distinguish tissue layers on Porites, though. We will have to work on that. And the POC tissue was miniscule, but that was expected from sectionining not going according to plan. Need to be super careful with POC slides when doing washes to not lose tissue.