This protocol has been optimized for 6 – 10 dpc embryos. Important points: Embryos should be dissected and quickly transferred to fixative as phosphorylation of MAPK is very labile. Fixation must be enough to preserve the diphosphorylation state of MAPK but not too much to prevent antibody recognition. All steps should be carried out at 4oC (except as noted). Secondary antibody must have low cross-reactivity to mouse tissues. Washing is very important – if using Alk-phos or HRP, it is best to take out a few embryos to stain and check to see if washing has been sufficient. It is always a good idea to include no 1o antibody controls.
1. Dissect embryos out in cold PBS, then fix in 8% paraformaldehyde overnight @ 4° C, with rocking
2. Wash in PBS containing 0.5% NP40 twice, 10 min. each
3. Dehydrate on ice through 25% methanol/75% PBS, 50% methanol/50% PBS, 75% methanol/25% PBS and 100% methanol, 15 min. for each step.
4. Bleach/block in methanol:30% hydrogen peroxide (5:1) for 1-2 hr @4°C, with rocking. 5. Wash in methanol (10 minutes).
6. Rehydrate on ice through 75% methanol/25% PBS, 50% methanol/50% PBS, 25% methanol/75% PBS, 100% PBS + 0.1% Triton X-100 15 min. each step.
7. Wash 2 times in PBSST (5% sera, 0.1% Triton in PBS) for1 hr @4°C, with rocking
8. Dilute phospho-MAPK Ab* in PBSST, incubate O/N @ 4°C, with rocking
*Diphosphorylated ERK 1 & 2(dpERK, clone MAPK-YT) mouse monoclonal Sigma M-8159 used at 1:250 dilution
or phosphorylated p44/p42 MAPK- rabbit polyclonal [Cell Signalling #9101] used at 1:350 dilution
9. Wash out antibody: 2x15 minutes @ room temp, then 5x1 hr+ @4°C with PBSST, with rocking.
10. Dilute secondary biotin donkey-antimouse antibody [Jackson #715-065-151] 1:250 in PBSST
OR biotin donkey-antirabbit [Jackson #711-065-152] 1:350 in PBSST and incubate O/N @4°C with rocking
11. Wash out antibody: 2x15 minutes @ room temp, then 5x1 hr+ @4°C with PBSST, with rocking.
12. Dilute CyX-streptavidin** (or alk-phos streptavidin) in PBSST, incubate O/N @4°C with rocking.
**Laura prefers Cy3-streptavidin from Jackson #016-160-084
OR for HRP staining:
mix 5 ul Vector reagent A + 5 ul Vector Reagent B in 1 ml PBSST; let sit for 15 minutes, then place embryos in buffer and incubate 3 hrs to O/N @4°C with rocking.
If using Alk-phos or HRP remove a few embryos, stain (SEE BELOW), and if necessary continue washing O/N.
If using fluorophore, monitor washing under fluorescence microscope.
13. If using hrp, stain with DAB (see below)
14. Clear in 50% glycerol/50% PBS
15. Clear in 70% glycerol/30% PBS
HRP DAB Staining:
Final wash in PBT (0.2% BSA, 0.1% Tween-20 in PBS)
For HRP detection, incubate embryos in 0.3 mg/ml DAB in PBT @RT for 20 minutes
Add H2)2 to 0.03% and incubate until color density looks good (2-10 minutes)
Rinse embryos 3-4 times with PBS, postfix in 8%PFA for 1 hour @ room temp.
Clear embryos... steps 14-15 above.
1. Dissect embryos out in cold PBS, then fix in 8% paraformaldehyde overnight @ 4° C, with rocking
2. Wash in PBS containing 0.5% NP40 twice, 10 min. each
3. Dehydrate on ice through 25% methanol/75% PBS, 50% methanol/50% PBS, 75% methanol/25% PBS and 100% methanol, 15 min. for each step.
4. Bleach/block in methanol:30% hydrogen peroxide (5:1) for 1-2 hr @4°C, with rocking. 5. Wash in methanol (10 minutes).
6. Rehydrate on ice through 75% methanol/25% PBS, 50% methanol/50% PBS, 25% methanol/75% PBS, 100% PBS + 0.1% Triton X-100 15 min. each step.
7. Wash 2 times in PBSST (5% sera, 0.1% Triton in PBS) for1 hr @4°C, with rocking
8. Dilute phospho-MAPK Ab* in PBSST, incubate O/N @ 4°C, with rocking
*Diphosphorylated ERK 1 & 2(dpERK, clone MAPK-YT) mouse monoclonal Sigma M-8159 used at 1:250 dilution
or phosphorylated p44/p42 MAPK- rabbit polyclonal [Cell Signalling #9101] used at 1:350 dilution
9. Wash out antibody: 2x15 minutes @ room temp, then 5x1 hr+ @4°C with PBSST, with rocking.
10. Dilute secondary biotin donkey-antimouse antibody [Jackson #715-065-151] 1:250 in PBSST
OR biotin donkey-antirabbit [Jackson #711-065-152] 1:350 in PBSST and incubate O/N @4°C with rocking
11. Wash out antibody: 2x15 minutes @ room temp, then 5x1 hr+ @4°C with PBSST, with rocking.
12. Dilute CyX-streptavidin** (or alk-phos streptavidin) in PBSST, incubate O/N @4°C with rocking.
**Laura prefers Cy3-streptavidin from Jackson #016-160-084
OR for HRP staining:
mix 5 ul Vector reagent A + 5 ul Vector Reagent B in 1 ml PBSST; let sit for 15 minutes, then place embryos in buffer and incubate 3 hrs to O/N @4°C with rocking.
If using Alk-phos or HRP remove a few embryos, stain (SEE BELOW), and if necessary continue washing O/N.
If using fluorophore, monitor washing under fluorescence microscope.
13. If using hrp, stain with DAB (see below)
14. Clear in 50% glycerol/50% PBS
15. Clear in 70% glycerol/30% PBS
HRP DAB Staining:
Final wash in PBT (0.2% BSA, 0.1% Tween-20 in PBS)
For HRP detection, incubate embryos in 0.3 mg/ml DAB in PBT @RT for 20 minutes
Add H2)2 to 0.03% and incubate until color density looks good (2-10 minutes)
Rinse embryos 3-4 times with PBS, postfix in 8%PFA for 1 hour @ room temp.
Clear embryos... steps 14-15 above.