Wholemount staining of embryos

• Fix embryos in formalin or MEMFA for one hour at room temperature with mixing. Rinse with TBS, replace with methanol, store at -20oC.
• Rehydrate by slowly adding TBS over a ten minute period. Once sample are in TBS, do 2 x 5' washes with TBS. Use haematology mixer (these are really good mixers Fisher cat#14-060-1)
• Block protein binding sites by incubating embryos in TBS plus 2 mg/ml BSA plus o.1% (v/v) triton-X-100 plus 20% heat inactivated normal serum (donkey or goat) at room temperature for 15 to 30'. Use nutator (Clay Adams, Fisher cat#14-062)
• Remove blocking solution and add primary antibody (500 ul), incubated overnight at 4 degC if sensitivity is not a problem. Antibody is added either as a cell culture supernatent, in which case it is used either neat or diluted with blocking solution, or as ascites or polyclonal in which case it is diluted in blocking solution. The dilution depends on the antibody. Ascites can often be diluted 1/2000 or more. Polyclonals depend on background levels, 1/500 and 1/100 are worth trying first. For affinity purified polyclonals try 1/20 and 1/50.
• Wash with at least five changes, 5 ml per wash, an hour each, of TBS plus 0.1% tween-20 (TBST) on hematology mixer.
• Block as above 15 to 30'.
• Secondary antibody is added in blocking solution. Concentration varies, but 1/500 works for most. For monoclonal primaries use anti-mouse coupled to AP or HRP, for polyclonals use anti-rabbit coupled AP or HRP. Incubate at 4 degC overnight on Nutator.
• Wash 5 times, 60' each, 5 ml per wash TBST
• add 2 mM levamisole to final wash if you are going to use alkaline phosphatase development
• stain in enzyme substrate

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