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- Performing immunostaining on cleavage-stage Helobdella embryo requires patience. It is advisable to extend antibody incubation to 4-10 days (depending on the location of the antigen; the deeper in the yolky blastomeres the longer it should take). It is also advisable to increase the frequency and length of the washing steps.
- In general, use 1% Triton X100 for the nuclear antigen. For cytoplasmic antigen, use 0.1% Tween-20.
- For late stage 9 and beyond (after coelom formation), immunostaining is best performed on the dissected germinal plates or the chitinase treated whole embryos. Chitinase treatment is carried out by incubating the embryo with 0.5 U/ml chitinase (Sigma C6137) in buffered saline at room temperature overnight. [Note: though leeches does not have chitin-containing cuticle, chitinase may help to loose up extracellular matrix by breaking down glycosidic bonds of the matrix molecules.]
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