By Yacine Graba, René Rezsohazy
Hox Genes: tools and Protocols explores strategies and methodologies which arose from or have been effectively utilized to the learn of Hox genes and Hox proteins, on the intersection of experimental embryology, genetics, biochemistry, body structure, evolutionary biology and different lifestyles sciences. This particular quantity starts off with a bit on discovery and sensible research of Hox genes after which it maintains onward to debate mode of motion and biomedical functions of Hox proteins. Written within the hugely profitable Methods in Molecular Biology sequence structure, chapters contain introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, with ease reproducible laboratory protocols and tips about troubleshooting and fending off identified pitfalls.
Expert and useful, Hox Genes: equipment and Protocols serves as an excellent consultant to researchers striving to maneuver ahead during this dynamic and fascinating quarter of study.
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4. 5 % Triton-X-100. 5 %) in a beaker and mix on a magnetic stirrer (see Note 7). 7 Immunofluorescence 1. Blocking buffer: add 10 % normal serum to PBS-T. Use serum from the same species in which the secondary antibody to be used was produced (see Note 8). Store at 4 °C until use. 2. Antibody dilution buffer: 1 % BSA in PBS-T. Mix well on a magnetic stirrer. Store at 4 °C until use (see Note 9). 3. Dako Pen (Dako). 4. Vectashield ® with DAPI (Vector laboratories). 5. Lab-Tek® chamber slide (Nunc™).
Pendleton JW, Nagai BK, Murtha MT et al (1993) Expansion of the Hox gene family and the evolution of chordates. Proc Natl Acad Sci U S A 90:6300–6304 7. Zhong Y-F, Holland PWH (2011) HomeoDB2: functional expansion of a comparative homeobox gene database for evolutionary developmental biology. Evol Dev 13:567–568 8. Bürglin TR (2011) Homeodomain subtypes and functional diversity. Subcell Biochem 52:95–122 9. Holland PWH, Booth HAF, Bruford EA (2007) Classification and nomenclature of all human homeobox genes.
While preparing the antibody dilution, keep the antibody vial on ice. If the antibody of interest has to be kept at −20 °C, sampling the antibody into smaller aliquots will avoid the antibody to undergo multiple temperature shifts. 47. Depending on the antibody, primary antibody incubation time could vary from 60 min to O/N. Incubation at 4 °C increases the specific antibody binding to its antigen. 48. Washing oocytes/embryos after primary/secondary antibody incubation is essential to discard the unbound antibodies.