http://www.stjuderesearch.org/depts/pathology/meadlab/docs/whc.docDAY 1
Note: use 5 ml of each solution per vial and incubate @ RT unless noted!
Vials are from Fisher, cat # 03-338 B
After sorting embryos by stages and putting them in vials (use 4 5 embryos for each stage you want to examine), fix and dehydrate them @ RT by adding the following:
1. 1 x MEMFA, tilt the vials slowly a few times
2. 1 x MEMFA, twice 30 min each on nutator
3. 25% EtOH, 5 min on nutator
4. 50% EtOH, 5 min on nutator
5. 75% EtOH, 5 min on nutator
6. 100% EtOH, 5 min on nutator, repeat twice
Important: Instead of adding actual dilutions (25, 50, and 75%) of EtOH, take a quarter of the last MEMFA volume out of vials, and fill them up with 100% EtOH. This will, essentially, be the same as washing embryos with 25% EtOH. When its time for 50% EtOH, simply take out yet another quarter of volume, and fill it up again with 100% EtOH this will be equal to washing with 50% EtOH. Do exactly the same for the 75% EtOH wash. When a quarter of the volume is aspirated for the fourth time, the vials will have ~ 100% EtOH.
Let the embryos stay in last 100% EtOH as long as possible before storing them @ - 20C at the end of the day
DAY 2
Allow embryos to warm to RT before continuing!
Rehydrate by washing in:
7. 100% EtOH, 5 min on nutator
8. 75% EtOH, 5 min on nutator
9. 50% EtOH, 5 min on nutator
10. 25% EtOH/75% MAB, 5 min on nutator
11. 100% MAB, 5 min on nutator, repeat three times
Important: Rehydrate embryos by performing the aforementioned dehydration technique in reverse. Use 1 x MAB to replace each aspirated quarter volume.
H2O2 sensitization and treatment:
12. Incubate in 5% Acetic Acid with 0.1 M K2Cr2O7, 30 min on nutator. This sensitizes them to H2O2 and destroys endogenous alkaline phosphatase.
13. Wash with MAB, 5 min on nutator, repeat three times
14. Treat with 5% H2O2 in MAB, 90 min on nutator
Important: Embryos of pre-tailbud stages tend to accumulate air bubbles on their surface when treated with H2O2. This leads to the embryos clumping up together and, thus, prevents their unexposed areas from being properly treated with 5% H2O2 in MAB. Tilting vials periodically a little faster than nutator speed should solve this.
15. Wash with MAB, 5 min on nutator, repeat three times
Primary
antibody incubation:
16. Block in BBT, 1 hr on nutator, repeat twice
17. Aspirate and replace with BBT + 5% Lamb Serum, 1 hr on nutator. Prepare with BBT left from the previous step. If the procedure involves too many vials, add only 2 ml, and incubate in upright position.
18. Incubate in Primary
antibody diluted according to specifications in MAB (usually, between 1:1000 and 1:10,000) overnight @ 4C
DAY 3
Washing and Secondary
antibody incubation:
19. Wash in BBT, 1 hr on nutator, repeat 4 times
20. Wash in BBT + 5% Lamb Serum, 1 hr on nutator. Prepare with BBT left from the previous step.
If the procedure involves too many vials, add only 2 ml, and incubate in upright position.
21. Incubate in Secondary
antibody (AP conjugated!) diluted according to specifications in MAB (the most common dilution is 1:1000), o/n @ 4C
Important: ensure compatibility between Primary and Secondary
antibodies!
DAY 4
Washing and detection:
22. Wash with BBT, 1 hr on nutator
23. Wash with MAB + 0.1% Tween 20, 1 hour on nutator, repeat 4 times
24. Incubate in Alkaline Phosphatase Buffer, 5 min on nutator, repeat twice
25. Replace buffer with 1 ml BM Purple, incubate upright on nutator. Should the reaction continue o/n, place the nutator @ 4C.
26. Stop the staining reaction by washing with MAB, 5 min on nutator, repeat twice
27. Fix with 1 x MEMFA for several hours
28. Gradually dehydrate by using the technique outlined in DAY 1
Solutions and reagents needed:
Alkaline Phosphatase Buffer 50 ml 100 ml
100 mM Tris Cl, pH 9.5 5 ml 1 M Tris Cl, pH 9.5 10 ml of the same
50 mM MgCl2 2.5 ml 1 M MgCl2 5 ml of the same
100 mM NaCl 1 ml 5 M NaCl 2 ml of the same
0.1% Tween 20 50 l 100 l of the same
BBT
MAB, 1% Bovine Albumin (Sigma cat # A-7906), and 0.1% Triton X-100
Lamb Serum
Thaw Lamb serum (Gibco BRL cat # 16070-096) o/n @ 4C
Heat-inactivate complement @ 56C for 30 min
Centrifuge @ 10,000 rpm for 20 min @ 4C to remove particulate material
Store @ - 20C in 25 ml aliquots
MAB (Maleic Acid Buffer), pH 7.5
100 mM Maleic acid 23.21 g
150 mM NaCl 17.53 g
Dissolve in approximately 1800 ml H2O, adjust pH to 7.5, volume to 2 L, and store @ RT
1 x MEMFA
5 ml 10 x MEMFA salts
5 ml 37% Formaldehyde (Fisher cat # F79-500)
40 ml H2O
Make fresh as needed do not store!
10 x MEMFA salts 500 ml
1 M MOPS 115.6 g
20 mM EGTA 3.8 g
10 mM MgSO4 1.23 g
Adjust pH to 7.4 with 10 M NaOH, filter-sterilize, and store @ 4C
The solution will turn yellow this is OK
BM Purple
BM Purple reagent from Roche, 50 ml, cat # 1 442 074, (stored @ 4C)
K2Cr2O7
Sigma cat # P 2588, 250 g
Secondary
antibodiesSecondary: Anti-Mouse IgG, AP conjugated, Sigma cat # A-3563
Anti-Rabbit IgG (Whole Molecule), AP conjugated, Sigma cat # A 8702
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