Fixing and decalcifying Pocillopora tissue for LCM, second test before summer experiments

Protocol here

Sectioning and performing LCM on the second branch that was prepared on 4/29/24

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
4. Day 1 (6/12/24): Section onto PEN membrane slide for LCM as well as extra slides for confocal morpholgy imaging and backup for RNA and DNA extraction
   1. All surfaces and equipment should be treated with RNAse cleaner!!!
   2. 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
   3. Prepare solutions:
      1. 70% Ethanol, 2 containers
      2. 100% Ethanol
   4. 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
   5. 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
         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!
   6. Freeze slides at -80 ºC
5. Day 2 (6/13/24)
   1. Notes: this protocol went super well but there was definitely still OCT. Might need longer 70% Ethanol rinse, maybe in a petri dish, after the cresyl violet.
      1. might need to do it with ice-cold RNase free water if the OCT is still there
      2. Otherwise, though, the tissue definitley stayed on much better than last time. And the PEN membrane slide was much easier to work with during LCM.
      3. Cutting and dropping the tissue into the collection vessel went much better than on 5/8. Only issues with cutting was when there was excess OCT.
   2. Morning of LCM, bring slide up to room temperature, slowly to avoid formation of water condensation inside the container
      • Thaw frozen slides at 4°C for 30 min.
      • 15 minutes at room temp in dessicator
        • Air dry, covered to prevent dust from accumulating
   3. LCM time!
      1. If possible, collect cells in lysis buffer, let lyse at RT for X mins before putting on dry ice and putting at -80 ºC
      2. Different Kit Options:
         1. Charm RNA - collect cells in 40 uL lysis buffer
            1. without BME - maybe add back in the lab?
            2. test with or without?
         2. Charm DNA - collect cells in 40 uL lysis buffer
         3. Zymo Quick RNA Microprep - collect cells in 40 uL RNA lysis buffer
            1. Based on this paper
            2. See extraction post here
         4. Zymo Quick DNA/RNA Microprep - collect cells in 40 uL DNA/RNA lysis buffer
            1. Collect cells in 20 uL DNA/RNA lysis buffer in tube cap
               1. Spin down at 800 rcf to ensure all cells made it down into the tube
               2. Add 20 uL more lysis buffer
               3. Vortex 5 seconds
               4. Transfer to DNA column and centrifuge
               5. Spin at 12,000 rcf for 30 seconds –> keep column with DNA, transfer flow-through to new tube
               6. Add 40 uL ethanol to flow through and mix well, spin down in RNA column
               7. DNase treatment on RNA column
               8. Both columns: Prep buffer, 400 uL
               9. Both columns: Two more washes, 700 uL and 400 uL
               10. Both columns: Elute in 6-15 uL nuclease free water
            2. proK digestion at some point??