Molecular Methods in Developmental Biology Molecular Methods in ...

10 Greentein factors are to be used in the medium, bovine serum albumin (BSA, Sigma)should be added to 0.1% w/v to block nonspecific protein binding.4. The agarose lining of dissection and incubation plates prevents sticking of explants.Depending on the number of caps to be assayed, it is essential to have sufficient numbersof dissection dishes, as they quickly become full of yolky debris during dissection.At least one 35-mm dish per 20 embryos/caps to be dissected is recommended.Depending on the number of conditions and caps to be assayed, agarose-lineddishes or multiwell plates must also be prepared for the caps after dissection. Acritical factor is that explants tend to fuse with one another, which can obscureobservation of morphological responses. Cap fusing has two effects. One is that,like rounding up, it excludes penetration or access of soluble factors. The other isthat scoring morphological changes is much harder in fused caps than in singlecaps. Where neither morphology nor factors in the medium are important, capfusion seems to have little effect on, for example, gene expression. To keepexplants separate, they can be assayed as one explant per well in an agarose-lined96-well tissue culture plate. Alternatively, two or more explants can be placed inseparate depressions made in agarose-lined dishes or larger wells. Depressionsare made using the sealed, red-hot end of a glass Pasteur pipet or metal fork.Alternatively, they can be cast into the agarose as follows. A mold is drilled in ablock of Teflon or similar material consisting of an array of 1.8-mm-diameter ×1.0-mm-deep depressions in the floor of a 4-mm-deep recess. The recess isslightly smaller that the Petri dish to be used for the embryos. A nonadhesivesilicone compound, such as Dow-Corning Sylgard 184, is cast in the mould togenerate a disk or square of rubber about 2 mm thick with 1-mm pimples on theunderside. This is floated on the surface of molten 1% agarose and removed afterthe agarose has set, leaving depressions suitable for embryos and explants.5. For straightforward morphological assays, such as elongation in response toactivin, as few as two caps per condition is sufficient and gives reproducible andquantitative results. For some morphological assays, such as for FGF, severalcaps are required because the morphological response is weaker and more unreliable.For RNA analysis by reverse transcriptase–polymerase chain reaction(RT-PCR), one or two caps per condition is minimally sufficient. However, morecaps will improve RNA yield per cap and enable duplicate assays for multiplegenes—strongly recommended for RT-PCR. For RNA analysis by RNase protectionassay (RPA), 10 caps per condition is advised. Although this seems likemore work, RPA enables several genes to be quantitatively analyzed in the sametube. This provides better quantitative control than with RT-PCR. Forwholemount in situ hybridization, the number of caps needed is largely a matterof taste, provided the gene expression is patently reproducible. Similarly, caps tobe harvested for immunostaining or conventional histological staining should benumerous enough to allow for some losses during workup and for persuasivereproducibility to be apparent. Generally speaking, it is better to cut additionalcaps than to economize. With practice, it should be possible for an average workerto dissect 60–100 caps per hour.