Pecam staining on whole Embryoid Bodies
Immunocytochemistry on mouse embryos
Immunocytochemistry on paraffin sections
Western blots
Immunoprecipitation
Benzidine staining for red blood cells
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Pecam staining on whole Embryoid bodies
1. Harvest ebs, rinse in PBS. Benzidine stain (optional). Fix overnight in 3.7% formaldehyde in PBS at 4 C. Note: do not dehydrate the ebs into methanol. This appears to quench the signal greatly. Also note: this protocol doesn't work on embryos. Embryos done this way give very high background.
2. Rinse ebs in PBS (CMF) 2-3X to get rid of fixative
3. Block all day at 4 C. in 3% milk, 0.1% Triton X-100 in PBS. Rock in microfuge tubes cold room or case. Store milk solutions at 4 C. so the milk won't spoil.
4. Dilute PECAM antibody (MEC 13.3 from Pharmingen) 1:500 in blocking solution. Add to ebs and let rock at 4 C. overnight.
5. In the morning, discard antibody solution and rinse ebs once with blocking solution. Then wash 5X for 1 hour each with blocking solution at 4C. on the rocker.
6. Dilute secondary antibody (anti-Rat-HRP from Sigma) 1:500 in blocking solution and add to ebs. Incubate overnight at 4 C. on rocker.
7. In the morning, discard the antibody solution and rinse ebs once with blocking solution, then wash 5X for 1 hour each in blocking solution at 4 C. on the rocker.
8. Rinse bods in 0.2% BSA in PBT (PBS + 0.1% Triton X-100). Then wash for 20 min in this soslution at room temp.
9. Prepare DAB solution by adding 1 tablet (10 mg) to 15 mls of PBT. It will take a few minutes for the tablet to dissolve. If desired, add nickel chloride to 0.5% to make stain dark blue instead of rust-colored. Mix well, then let heavy particulate settle. Soak bods in this solution for 20 minutes.
10. To remaining DAB solution add 1 ul of 30% hydrogen peroxide per 1 ml of DAB solution. Add to bods. Color should develop almost instantly. Let go for 1-5 minutes, then remove DAB solution, rinse in PBS, and postfix overnight in 3.7% formaldehyde. Note: if you use nickel chloride you must dehydrate and coverslip in permount otherwise the stain will leach out.
Note: This works well for smooth muscle actin also.
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Benzidine Staining to reveal red blood cells
Benzidine (100 mg in safety pac, B-1883 from Sigma)
Stock solution: Inject 20 ml of 0.5 M Acetic Acid into the 100 mg bottle of Benzidine powder. Store solution in the dark at room temp. Keeps for months.
Prepare working solution just before use: Withdraw 1 ml benzidine stock solution using a syringe and 28 g needle. Squirt into eppendorf tube and add 20 ul 30% H2O2.
Dilute this 1:4 in PBS (CMF). A precipitate may form but this is not fatal.
Rinse embryos or embryoid bodies in PBS. Withdraw most of the buffer and add about 1 ml of the staining solution. Use transfer pipet to transfer embryoid bodies to 35 mm petri dish to observe in dissecting scope. It may be easier to use tweezers to transfer embryos to staining solution which is already in petri dish. Blue color may come up in a minute or so and staining is usually complete in about 10 minutes.
Rinse, fix, photograph, embed in paraffin, whatever.
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