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Histology Protocol

Fix:                                                                  Polybed:

1 mL 0.2 M cacodylic acid                               9.6 mL of Polybed

400 µL water                                                    6.2 mL of DDSA

400 µL 10% ultrapure formaldehyde                 4.2 mL of NMA (mix well before adding phenol)

200 µL 25% gluteraldehyde                              400 µL of T-phenol

Fix fry overnight (O/N) at 4°C (no longer than O/N).  If necessary, place fry in 5% sucrose/PBS after one night of fixation until washes.

Washes:

Solution	# of washes	Time
1. 0.2M cacodylic acid	3 x	5 minutes each
2. water	3 x	5 minutes each
3. 50 % EtOH	2 x	5 minutes each
4. 70 % EtOH	2 x	5 minutes each
5. 80 % EtOH	2 x	5 minutes each
6. 95% EtOH	2 x	5 minutes each
7. 100 % EtOH	2 x	10 minutes each
8. Xylene / EtOH (1:1)	1 x	30 minutes
9. Xylene	1 x	30 minutes
10. Polybed / Xylene (1:1)	1 x	60 minutes
11. Xylene / Polybed (1:2)	O/N
Total Time:		3.5 hours

The next day, place fry in mold and add fresh polybed (100%) for 60 minutes.  Transfer mold to 37°C O/N. 

The next day, check orientation of larva (you can use a needle and microscope to position larva within mold) and transfer to 42°C for 8 hrs.  After 8 hrs, check orientation once more, and transfer mold to 60°C for 48-72 hrs.