Solutions

1) Calcium- and Mg^++-free PBS

2) Paraformaldehyde (PFA) (Polysciences)

3) Methanol

4) 30% Hydrogen Peroxide (H₂0₂) (Sigma)

5) Powdered Milk

6) TritonX – 100 (Boehringer Mannheim)

7) PBS / 0.1% TX-100/0.2% BSA

8) Benzyl alcohol

Equipment and Supplies

9) Glass scintillation vials, one for each embryo. (Do not use polysterene plastic as the benzyl alcohol will dissolve the plastic.)

10) Anti-mouse HRP (Jackson Immuno Research cat#: 115-035-146)

11) NiCl(0.5% w/v ) (Sigma)

12) DAB (Di-aminobenzidine)(1 Tablet (10 mg)(Sigma, cat# D5905)

Day 1:

Dissect embryos in PBS and fix overnight in 4% PFA + PBS using a glass scintillation vial.

Day 2:

The following day begin by washing the embryos for 10 to 20 minutes in PBS (wash steps dependend on the age of the embryos). For larger embryos use the longer time.

They should then be dehydrated by sequentially emersion and shaking in the following solutions:

10 – 20’ in 25% MEOH / PBS

10 – 20’ in 50% MEOH / PBS

10 – 20’ in 80% MEOH / PBS

10 – 20’ in 100% MEOH

Following dehydration, the embryos can be stored at – 20 °C for at least up to 6 months

Staining procedure (Day3)

Block endogenous peroxidase activity for 4 – 5 hours in 100% Methanol with 5% H₂O₂ (stock is 30%) shaking at room temperature

Rehydration

Rehydrate embryos by sequential emersion and shaking in the following solutions

80% MEOH/PBS 10-15’

50% MEOH/PBS 10-15’

25% MEOH/PBS 10-15’

PBS PBS 10-15’

Block unspecific antibody binding by 2 incubations for 1 hour each at r.t. or overnight at 4°C in 3% MILK in PBS 0.1% TritonX – 100.

Stain at 4°C overnight with a 1:100 dilution of the 2H3 monoclonal antibody (Developmental Studies Hybridoma Bank , supernatant) in 3% MILK ,PBS / 0.1% TX – 100.

Day 4:

Wash the embryos 5–6 times for 1hr. each in 3% MILK PBS / 0.1% / TX-100 at 4°C

Stain overnight at 4°C using anti-mouse HRP (Jackson Immuno Research) diluted 1:500

Wash the embryos 5–6 times for 1hr. each in 3% MILK PBS / 0.1% TX-100

Wash the embryos 20 min. in PBS / 0.1% TX-100/0.2% BSA

at same time make up DAB (1Tablet (Sigma 10 mg) in 33 ml in PBS / 0.1% TX-100/0.2% BSA (Final concentration 0.3mg / ml). When well dissolved (It takes about 30 min or longer rotating) add NiCl to 0.5% w/v final concentration (167mg/33mls). Add 2ml to each embryo, then incubate 20 min at room temperature shaking, then add 0.03% H₂O₂% (1:1000 dilution of 30% stock) for 5 – 10 min (watch the color of the embryos as they incubate with constant gentle shaking). Stop the reaction when the blue color is well developed by washing in PBS 0.1% Triton X100.

Post fix in 2% PFA, 0.1% gluteraldehyde overnight at 4°

Day 5

Dehydrate the embryos by sequential washes for 15 min. each at room temperature in :

25% MEOH/PBS

50% MEOH / PBS

80% MEOH / PBS

100% MEOH

Clear the embryos in benzyl alcohol.