Dechorionate embryos, if needed.

Fix embryos in 500µL4% paraformaldehyde for 2h 0m 0s at Room temperature or overnight at 4°C.

Wash in 1mL100% MeOH at Room temperature for 0h 10m 0s. (1/3)

Wash in 1mL100% MeOH at Room temperature for 0h 10m 0s. (2/3)

Wash in 1mL100% MeOH at Room temperature for 0h 10m 0s. (3/3)

Store at -20°Clong-term (can be months or longer)

Wear gloves and treat surfaces for RNAses.

Rehydrate the embryos

Wash embryos in 0.5mL 75% Methanol/25% PBTween, rocking, for 0h 5m 0s at Room temperature in 1.5 mL centrifuge tubes.

Wash embryos in 0.5mL 50% MeOH / 50% PBTween, rocking, for 0h 5m 0s at Room temperature

Wash embryos in 0.5mL 25% MeOH / 75% PBTween, rocking, for 0h 5m 0s at Room temperature

Wash embryos in 0.5mL PBTween, rocking, for 0h 5m 0s at Room temperature (1/3)

Wash embryos in 0.5mL PBTween, rocking, for 0h 5m 0s at Room temperature (2/3)

Wash embryos in 0.5mL PBTween, rocking, for 0h 5m 0s at Room temperature (3/3)

Optional: Bleach embryos in0.5mLfreshly-made 3% H₂O₂ + 1.79millimolar (mM)KOH for up to 0h 5m 0s. Leave the tube caps open and monitor bleaching.

Rinse in 0.5mL PBTween (1/2)

Rinse in 0.5mL PBTween (2/2)

Permeabilize tissue. Option 1: proteinase K - proceed to step 6.1. Option 2: acetone - proceed directly to step 6.3.

Option 1: Digest with 1mL 10µg Proteinase K in PBTween at Room temperature for 0h 5m 0s or 0h 20m 0s or 0h 30m 0s . Proceed to step 6.2

Refix tissue in 0.5mL 4% PFA, rocking, at Room temperature for 0h 20m 0s . Skip to step 6.4.

Option 2: Incubate in 1mL 80% acetone/ 20% diH₂O at Room temperature for 0h 20m 0s.

Wash in 0.5mL PBTween, rocking, at Room temperature for 0h 5m 0s (1/3)

Wash in 0.5mL PBTween, rocking, at Room temperature for 0h 5m 0s (2/3)

Wash in 0.5mL PBTween, rocking, at Room temperature for 0h 5m 0s (3/3)

Separate embryos into designated tubes (example: sense vs. anti-sense tubes) if not done previously.

Incubate in 250µL prehybe in hybe oven set to 65°C, rocking, for at least 4h 0m 0s

Incubate with (0.1-1μg/mL) probe diluted in 250µL warmed prehybe , 65°C, rocking.

Remove probes. Probes can be stored at -20°C and reused up to 3 times.

Post-hybridization washes

Wash in 0.5mL 100 % (50% 5x SSC / 50% formamide) for 0h 10m 0s at 75°C rocking

Wash in 0.5mL 25% 0.2x SSC / 75% PBTween for 0h 10m 0s at 65Room temperature rocking

Wash in 0.5mL 100% PBTween for 0h 10m 0s at 65Room temperature rocking

Wash in 0.5mL 75% (50% 5x SSC / 50% formamide) / 25% 2x SSC for 0h 10m 0s at 75°C rocking

Wash in 0.5mL 50% (50% 5x SSC / 50% formamide) / 50% 2x SSC for 0h 10m 0s at 75°C rocking

Wash in 0.5mL 25% (50% 5x SSC / 50% formamide) / 75% 2x SSC for 0h 10m 0s at 75°C rocking

Wash in 0.5mL 2x SSC for 0h 10m 0s at 75°C rocking

Wash in 0.5mL 0.2x SSC for 0h 15m 0s at 75°C rocking

Wash in 0.5mL 0.2x SSC for 0h 15m 0s at 75°C rocking

Wash in 0.5mL 75% 0.2x SSC / 25% PBTween for 0h 10m 0s at 65Room temperature rocking

Wash in 0.5mL 50% 0.2x SSC / 50% PBTween for 0h 10m 0s at 65Room temperature rocking

Incubate in 0.5mLblock for at least 2h 0m 0s Room temperature , rocking

Incubate 4h 0m 0s``4°Cwith 0.5mL 1:5000 sheep AP-conjugated anti-DIG Fab fragments (or 1:2000 sheep AP-conjugated anti-FLU Fab fragments)

Remove antibody. Antibody can be stored at 4°C and reused up to 3 times.

Post-antibody washes

Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (1/10)

Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (10/10)

Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (2/10)

Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (3/10)

Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (4/10)

Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (5/10)

Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (6/10)

Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (7/10)

Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (8/10)

Wash in 0.5mL PBTween for 0h 10m 0sat Room temperature, rocking (9/10)

Make fresh NTMT buffer. Mix 1mL 1Molarity (M)Tris 9.5, 200µL``5Molarity (M)NaCl, 500µL``1Molarity (M) MgCl₂, 50µLTween20 and 8.25mLwater

Equilibrate embryos in 0.5mLNTMT buffer for 0h 5m 0sat Room temperature(1/2)

Equilibrate embryos in 0.5mLNTMT buffer for 0h 5m 0sat Room temperature(2/2)

Transfer embryos to multiwell culture plate (keep the tubes)

Wash in 1mL NTMT buffer for 0h 5m 0sat Room temperature

Prepare fresh stain solution.

Add 4.5µL NBT and 3.5µL BCIP to NTMT buffer. Protect from light.

Replace NTMT in culture plates with 1mL of the freshly prepared NBT/BCIP stain solution.

Cover with foil

Stain in the dark until staining reaches desired intensity, typically when color begins to appear in the sense controls. This step can last hours to days.

Stop the reaction by rinsing in 2mL PBTween

Fix tissue after staining

Transfer embryos back to their tubes

Fix embryos in 0.5mL 4% PFA, rocking, at Room temperaturefor 0h 20m 0s

Wash in 0.5mLPBTween, rocking, at Room temperaturefor 0h 5m 0s(1/3)

Wash in 0.5mLPBTween, rocking, at Room temperaturefor 0h 5m 0s(2/3)

Wash in 0.5mLPBTween, rocking, at Room temperaturefor 0h 5m 0s(3/3)

Prepare embryos for glycerol imaging

Wash embryos in 1mL 30% glycerol / 70% PBTween at Room temperature for 0h 10m 0s while rocking and covered in foil.

Wash embryos in 1mL 50% glycerol / 50% PBTween at Room temperature for 0h 10m 0s while rocking and covered in foil.

Wash embryos in 1mL 80% glycerol / 20% PBTween at Room temperature for 0h 10m 0s while rocking and covered in foil.