Day 1

Xylene clearing ~2 hrs

1. Rock embryos in 1:1 MeOH:EtOH 5 min (Waste -> organic waste bottle)
2. Rinse 2x with EtOH
3. Rock in EtOH 5 min
4. Remove all but 100 uL EtOH,
5. Add 900 uL Xylenes 1.0 hr (embryos should go transparent)
6. Rinse 2x EtOH (embryos should all turn white again)
7. Rock in EtOH 5 min

Post-fix ~45 min

1. Rinse 2x MeOH
2. Rock in MeOH 5 min
3. Rock in 1:1 MeOH: ½x PBT, 5% F 5 min (Waste -> formaldehyde waste bottle)
4. Rinse in 1/2x PBT, 5% Formaldehyde (F)
5. Rock in 1/2x PBT, 5% F 25 min exactly

Prehyb ~3 hrs

1. (Hyb Solution (HS) is in -20 C)
2. Rinse 2x with PBT (Waste -> aqueous waste vacuum)
3. Rock 4x in PBT 15 min (HS waste -> Formaldehyde waste)
4. Rock in 1:1 PBT: HS 10 min room temp
5. Rinse 2x with HS 10 min 55 C
6. Rock 3x in HS over 1.5 hrs during 1.5hrs 55 C
7. (Use 55C Eppendorf shaker next to miniprep bench)

Probe ~15 min prep, O/N incubation

1. In a clean tube,
2. Add ~4uL of each probe to 200 uL of HS
3. Denature secondary structure. Heat 2-3 min 80-85 C
4. Transfer to ice 1-2 min 0 C
5. Remove hyb from embryos, Add probe 18-24 hr 55 C

Day 2

Hot Wash ~4 hrs

1. Pre-warm HS to 55 C 15 min / 50 mL keep warm for washes
2. Rinse 2x with HS 10 min Give 5 min to settle
3. Hot wash embryos in HS 4x + during 2 hrs + 55 C, rock occasionally
4. Rock in 1:1 PBT: HS 10 min Everything now at room temp
5. Wash 3x in PBT during 1 hr

Primary Antibody ~1hr, O/N incubation

1. Prepare Block: dilute Roche Blocking reagent 1:5 in PBT (this can be stored at 4C)
2. Rock in Block 45 min (waste -> vacuum)
3. Remove Block,
4. Add 1.25 uL of each antibody* to
5. 500 mL block per tube.
6. Rock overnight in antibody in cold room 14 hrs+ 4 C
7.  *(anti-Dig, anti-DNP, anti-Bio, anti-Fluorescein. Record source animal: rat, rabbit, goat…)  Roche sheep anti-dig (Roche purchase page)
   Roche mouse anti-bio. Abcam mouse anti-bio
   Roche(?) rabbit anti-DNP

Day 3

Wash out primary antibody ~ 1+ hr

1. Rinse 2x with PBT
2. Wash 5x in PBT during 1 hr +

Secondary antibody stain and rinse (in Dark!) ~ 5 hrs

1. Block 1 hr
2. 2ndary Alexa antibody 1:500 in block* 1.5 hrs (cover in foil)
3. Rinse 2x with PBT
4. Wash 6x with PBT during 2 hrs + (cover in foil)

Small molecule labeling

1. As desired, label with one or more of the following
2. WGA at 1:500 in PBT Rock 1 hr
3. Hoechst at 1:2000 in PBT Rock 20 min
4. Rock with Draq5 1:3000 in PBT Rock 20 min (cover in foil)
5. Wash 1x in PBT Rock 10-20 min

Mounting ~ 1hr

1. With a cut-off P200 tip, apply embryos to clean glass slide (~100-130uL)
2. Aspirate remaining liquid, be careful not to aspirate embryos
3. Apply 100-130 uL of room temp. mounting media (ProLong Gold, store at –20C)
4. Drag a P200 tip around to cover all embryos in mounting media
5. Slowly lower coverslip over embryos from one end. Can apply extra mounting media around edge to seal bubbles.
6. Seal with nail polish if desired.

(Colorimetric)

Colorimetric Reaction 0.5-4.5 hrs

1. Wash in staining buffer 2x 5 min (make fresh buffer!)
2. Remove buffer, add 1 mL staining soln
3. Using a cutoff P200 tip, Transfer to staining dishes, monitor 2 min-4hr (cover with box)

Stop Reaction ~1.5 hrs

1. Transfer embryos to a new eppendorf
2. Rinse 2x with PBT
3. Rock 1x in PBT 5 min
4. Rock 1x in 1:1 PBT:EtOH 5 min
5. Rock 8x in new EtOH 1 hr +

Mounting ~ 1 hr

1. Get clean slides and coverslips
2. Aspirate off EtOH, Add 1 mL xylenes 1-3 min (do not exceed 3 min, do not invert)
3. Remove xylene.
4. Add 300-400uL Permount mounting media (Fisher)
5. Mount embryos with a cutoff tip
6. Add cover slip, can wick media in from side
7. Store flat for at least the first year