Plastic Embedding and Sectioning of Xenopus laevis Embryos

Journal of Visualized Experiments www.jove.com Video Article Plastic Embedding and Sectioning of Xenopus laevis Embryos Souichi Ogata 1 , Shimako Kawauchi 1 , Anne Calof 2 , Ken W.Y. Cho 1 1 Department of Developmental and Cell Biology, University of California, Irvine 2 University of California, Irvine Correspondence to: Ken W.Y. Cho at kwcho@uci.edu URL: http://www.jove.com/index/Details.stp?ID=188 DOI: 10.3791/188 Citation: Ogata S., Kawauchi S., Calof A., Cho K.W.Y. (2007). Plastic Embedding and Sectioning of Xenopus laevis Embryos. JoVE. 3. http://www.jove.com/index/Details.stp?ID=188, doi: 10.3791/188 Abstract Protocol Fixation 1. Transfer whole embryos or dissected explants into MEMFA or 3.7% FA+PBS in a small glass scintillation vial (Fisher cat# 03-339-25B). Incubate vial on the nutator at RT, only for 2 hours, and not more than that. Do not leave the embryo in FA for more than 2 hours. Longer incubation in FA will make the embryonic tissue harder and result in a poor penetration of antibodies in the detection process. 2. Aspirate most of FA and add Dent's fixative up to the top of the vial (~ 4.5ml). Gently invert the vial for a few times to wash the embryos in Dent's, exchange with another 4.5ml of fresh Dent's, and incubate it in -20oC for at least 48 hours. Overnight is NOT enough. This step makes the embryos more permeable for the antibody. Embryos can be kept in this condition for several weeks. Immunofluorescence 1. Rehydrate the specimen. Incubate it in a graded series of methanol, as follows: 75% MeOH/H 2 O 10min 50% MeOH/PBS 10min 25% MeOH/PBS 10min PBS 10min PBS 10min 2. Make 1.5% agarose/PBS plates with 60mm plastic dishes. Pour PBS on top of the agarose after it has become solid. 3. Hemi-sectioning of the embryos: Put the embryos in a 1.5% agarose/PBS plate. Grab an embryo with a pair of forceps and cut it in half through the center of the embryo using a thin razor blade. Choose the ones that you have successfully cut in half with a straight cutting plane. 4. Transfer the bisected embryos into a glass scintillation vial with PBT. Exchange with 4.5ml of 20% goat serum+PBT. Incubate at RT for 2 hours on a nutator (Blocking). 5. Prepare an appropriate dilution of the primary antibody in PBT. 0.5ml of this per samples is needed. For instance, I used 1/200 dilution of rabbit anti-C-cadherin pAb to study the subcellular localization of C-cadherin in 5mm plastic sections. 6. Aspirate the blocking solution and add 0.5ml of the diluted primary antibody. Put the vials in stand-up position on a Styrofoam tube holder. Incubate overnight at 4oC on a nutator. 7. Remove the diluted primary antibody (this diluted primary antibody can be saved and reused in the next 1-2 weeks). Wash with 4.5ml of PBT for 4 times (40-60 minutes each) at RT. 8. Exchange with 0.5ml of 1/100 dilution of Biotin-conjugated secondary antibody in PBT. Incubate overnight at 4oC on a nutator. Cover the vials with aluminum foil to protect biotin from light. 9. From this point, care should be taken to protect the sample from light, i.e., keep the vials under some cover or aluminum foil. 10. Remove the Biotin-secondary antibody (this can be also reused in the next 1-2 weeks). Wash with or 4.5ml of PBT for 4 times (40-60 minutes each) at RT. 11. Exchange with 0.5ml of 1/200 dilution of fluorescently-labeled (FITC, Texas-red, etc.) Streptavidin in PBT. Incubate overnight at 4oC on a nutator. Copyright © 2007 Journal of Visualized Experiments Page 1 of 2