This protocol was adapted from the protocol of Rachel Bober (RachelBober) written on 10.12.2024.

1. Fixation

Copied from the protocol, not tested

1. 1.5 ml FSW (0.2 microns filtered seawater) in a 15 ml tube (for small fragments).
2. Cut the sample into approximately 2 cm fragments and place them in the tube.
3. Leave the fragments in the tube with the FSW for 10 min before adding 1M mgcl2.
4. Add 0.3 ml of 1M mgcl2 to a single tube, and cover the plate with foil for 10 min.
5. Remove the FSW from the tube.
6. Add 4% formaldehyde solution into the tube.
7. Second option - leave the 1.8 ml of FSW and 1M mgcl2 solution in the tube and add 0.5 ml of 16% Formaldehyde to the tube for a final concentration of 4%.
8. Leave at 4°C overnight, or 1-2 hr at room temperature.
9. Wash X3 with sterile 1x PBS solution.
10. For extended storage, store in 70% sterile ethanol, or leave in PBS for short storage of 2-3 days.

2. Decalcification

1. Wash X3 with PBS in RT, 15 min each.
2. For scleractinian- decalcification of the skeleton with 1:1 formic acid (50% in DDW) & Sodium citrate 20% (20% in DDW). Transfer the fixed sample into 50 ml tubes and add the decalcification solution (10 ml in each tube - for samples bigger than 3 cm add 15 ml) for 4-5 hours\overnight.
3. Transfer decalcified tissue into a cassete, mark the sample name on it using a crayon.
4. Wash X3 with PBS in RT, 15 min each. Start the dehydratation immediately.

3. Dehydratation and embedding

1. Preheat the incubator and three glass jars filled with paraffin to 58-60 °C for 4 h/overnight to ensure the complete melting of paraffin.
2. Dehydrate the tissue through a series of graded ethanol (with ddH2O) 50%, 75%, 90% and 100%, 15 min each. If the tissue is more than 2mm thick extend the last step (100%) to 30min.
3. Clear the tissue through a series of graded butanol (with ethanol) 50%, 75%, 100% and 100%, 30 min each (the last one can be extended to 1 h).
4. Remove the butanol and insert the tissue into a glass vial with melted paraffin, incubate 1h in the oven preheated to 58-60 °C
5. Transfer the tissue into another new glass vial with melted paraffin, incubate overnight
6. Transfer the tissue into the other glass vial with melted paraffin for 2h.
7. Take the cassete with the tissue out of the glass vial and use the embedding center to make the paraffin block out of it.
8. Let the block chill on ice for a few hours and then transfer to 40c overnight.

Save paraffin blocks at RT (in case of extra heated places it is better to store the block inside a box in a minus 40c fridge).Before sectioning in microtome, chill the blocks on ice or in ice-cold water.