Flash freezing fragments from three species from CBLS wetlab

1. Pocillopora - genotype X, fragment 1

1. Porites - genotype D

1. Montipora - genotype E

Protocol: Using sterilized forcepts, flash frozen in liquid nitrogen, then placed in 1.5mL tubes labelled:

• FF_POC_1
• FF_POC_2
• FF_POC_3
• FF_MON_1
• FF_MON_2
• FF_MON_3
• FF_POR_1
• FF_POR_2
• FF_POR_3

These tubes were parafilmed and put into zip lock bags and stored at -80 ºC until shipment on dry ice to facility.

Fixing and decalcifying fragments from three species from CBLS wetlab

Montipora capitata, Porites compressa, and Pocillopora acuta ( all collected from HI in October 2023 and kept in BT1/2 in CBLS wetlab since)

Protocol here

1. Fixing in three separate 5mL containers for good tissue:fixative ratio.
   • Day 1 (01/29/24): Fixed tissue in PAXgene fixative
     • sterilized, RNAse cleaned clippers and forceps used for all steps, cleaned in between species.
     • fixed in 5 mL PAXgene fixatives in 5mL tubes
   • Day 2 (01/30/24): Replaced fixative with stabilizer, transferred to 4 ºC
   • Day 3 (01/31/24): Started decalcification, kept on shaker in cold room (4 ºC)
   • Day 4 (02/01/24): Changed EDTA solution (24 hours)
   • Day 5 (02/02/24): Changed EDTA solution (48 hours) - POR might be done at this point
   • Day 6 (02/03/24): Changed EDTA solution (72 hours)
   • Day 7 (02/04/24): Decalc done after 96 hours. Tissue washed (10 mins, 1X RNAse-free PBS). Transferred into Paxgene tissue stabilizer for 24 hours at 4 ºC.
   • Shipping: 2/5/2024 (Day 8), on dry ice.

Image before decalcification:

Images after decalcification:

Pocillopora acuta

Montipora capitata

Porites compressa

box with decalc and FF samples sent on 2/5/24, delivered in NY on 2/6/24