Whole mount embryo staining for Tsp4b

FIXATIVE – ACETIC ACID- METHANOL for Tsp4b

For 100ml of fixative

Methanol – 95ml of 100% methanol
Glacial acetic acid – 5ml

Embryos of chosen stage (n = 25 – 50) are fixed for 4 hrs. in acetic acid-methanol fixative at -20°C. (Can also be left over night)
The embryos are re hydrated with PBT (PBS + 0.5% Triton + 2% DMSO+ 0.05% NaZ). Note: The concentration of Triton can be varied to suit your staining.
1. 1 X wash – 2min
2. 2X washes – each 10 min
Depigmentation: If the embryos have pigments, depigment them using H₂O₂/KOH solution.
1. 1% KOH - 3% H₂O₂ treatment. (100ul 10% KOH and 100ul 30% H₂O₂ in 800ul of water) – this takes between 20 mins to 30 mins. If the eyes are light brown in color, you can stop depigmentation.
2. Discard the depigmentation solution carefully using a pipette (sometimes embryos are floating in the foam)
3. Quickly rinse the embryos once withwater.
Incubate the embryos in cold acetone for 20 minutes at -20 °C The incubation timing appears to be critical.
Quickly rinse the embryos with water once. Allow the embryos to settle down by gravity on the tube stand. The eyes often appear engorged in older embryos due to osmotic shock. Remove as much of water as possible with a pipette without disturbing the embryos and replace with PBT immediately.
Rehydrate the embryos with PBT with 3 washes. Each wash for 10 minutes.
Add primary antibody cocktail dilution in PBT. (In some cases, the primary antibody is diluted in 10% NGS to reduce background. However, in my experience it hasn’t been an advantage. For Thbs4b, the antibody concentration is 1:1000)
The staining is left to proceed at Room Temperature or at 4°C for at least 12 hours or overnight.

DAY 2

Save the primary antibody dilution in a separate tube at 4°C. The solution remains good for at least 6 months.
Wash the embryos with PBT for 4 – 6 times. Each wash for 10 minutes.
Prepare a secondary antibody cocktail.
For fluorescence staining, choose the proper fluorophore tagged antibody. In the lab we currently have Cy3, TRITC, Texas red and Alexa 596 (red), FITC, Alexa488 (Green) and Cy5, (blue) tagged antibodies.
Prepare a dilution of 1:1000 of these antibodies in PBT and add to the embryos. For single antibody staining I choose Cy3 as the preferred fluorophore.
The staining proceeds for 2 hours at 4°C or room temperature. (I have not seen any advantage at 4°C compared to room temperature)
The antibody staining solution is discarded and the embryos are washed with PBT for 4 – 6 times.
Check for staining under the UV scope and proceed for Glycerol clarification.
The embryos are transferred to 75% – 80% glycerol/PBT solution and left overnight in this solution before mounting on slides.