Applications include assessment of gene expression patterns of novel genes (e.g.1,2, as well as changes in gene expression following insult (embryonic manipulations3, beads4, and electroporation of RNA or DNA constructs5). == Disclosures == The authors have nothing to disclose. == Acknowledgments == This work was supported by the Margaret M. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented. Keywords:Developmental Biology, Issue 20, whole mount in situ hybridization, gene expression, chick embryo Download video stream. == Protocol == == Part 1: Fixing the embryos == Fill dissection dish with ice cold depc-PBS. Keep on ice. Open the egg by tapping the shell with forceps and remove shell pieces. Remove thick albumin with forceps. Tilt yolk sac with coarse forceps so that embryo faces upwards. Using scissors, cut a square of yolk sac around the embryo. Using spoon, remove embryo from yolk and place on ice cold depc-PBS. Under the microscope, remove membranes and yolk and transfer to fixation dish. Pin down, remove depc-PBS. Replace with 4% PFA in depc-PBS and fix O/N at 4C. Remove fixative. Replace with ice cold depc-PBS. Using a blunt end microcapillary L-2-Hydroxyglutaric acid needle or a microdissection knife, perforate the nervous system and the heart cavities, to prevent trapping of probe. Wash 2x 10 mn depc-PBS with 0.1% Tween-20 (PBT). Dehydrate 10 mn each in 25%, 50%, 75% methanol in PBT. Dehydrate embryos twice in 100% methanol. Store at -20C in methanol. == Part 2: Prehybridization == Rehydrate embryos 10 mn each in 75%, 50% and 25% methanol in PBT. Wash 2x 10 mn in PBT. In the mean time, prepare snow chilly fixative (4% paraformaldehyde in PBT). Replace PBT with proteinase K remedy (3 ml/vial; final [5?g/ml] in PBT). Make sure the entire vial is exposed to proteinase K remedy by gently rolling the vial; observe Troubleshooting for Rabbit polyclonal to Zyxin exposure instances. Using RNase free Pasteur pipette remove proteinase K and add fixative (2 ml). Immediately place on snow for precisely 20 mn. All RT washes are performed on nutator. Wash 2x 10 mn in PBT, and 1x 10mn in PBT comprising 50% hybridization buffer. Wash 1x in hybridization buffer. Incubate at L-2-Hydroxyglutaric acid 65C in 2 ml hybridization buffer for 4-5 hr. == Part 3: Hybridize embryos with probes == DIG and FITC coupled probe synthesis is definitely explained in Appendix. The probe expected to give a strong signal, should be synhesized with FITC-containing nucleotides; the weaker probe should be synthesized with with DIG-containing nucleotides. Prewarm probes in hybridization buffer (500 ng/ml each) using a 2 ml vial. Promptly replace the hybridization buffer with the probe remedy. Do not let the embryos dry. Incubate for 16 hr at 65C. Replace probe with hybridization buffer, 2 washes, 30 mn each at 65C. Wash 15 mn in hybridization buffer/MABT (1/1 v/v/) at 65C. == Part 4: DIG antibody incubation and color reaction == Wash 2×20 mn in MABT. Wash 1 hr in 2% BBR/MABT. Wash 5 hr in 2% BBR/20%HISS/MABT (obstructing buffer). Incubate in 1:2000 DIG antibody diluted in obstructing buffer, O/N at 4C on nutator. Wash 3x 10 mn each in MABT, and 3x 1 hr each in MABT. Wash 2x 20 mn in NTMT. Add 200ul NBT/BCIP substrate to 10 ml NTMT. Immediately remove NTMT from vial and change with 1-2 ml NBT/BCIP buffer. Place in dark. Monitor color reaction after 20 mn, and at 20 mn intervals thereafter. Following color reaction (should appear blue), wash in PBS for 10 mn, pin down on fixation dish filled with PBS, and replace remedy with 4% PFA in PBS. Fix O/N at 4C. Transfer to fresh scintillation vial. Wash in PBST (PBS with 1% Tween-20) 2×10 mn. Wash in MABT 2x 10 mn. Incubate in MABT 30 mn at 63C. == Part 5: FITC antibody incubation and color reaction == Wash 2x 10 mn in MABT, 1 hr L-2-Hydroxyglutaric acid in 2%BBR/MABT, 5 hr in obstructing buffer Incubate in alkaline phosphatase-coupled anti-FITC-antibody (1:500) O/N at 4C. Wash 3x 10 mn each in MABT, and 3x 1 hr each in MABT. Wash in 2x 20 mn in TBS pH 8.45 with 0.1% Tween-20 (TBST). Prepare Vector Red working remedy (5 ml) in TBST using reagents 1,2 and 3 offered in kit. Immediately remove TBST from vial and change with Vector Red buffer. Place in dark. Monitor color reaction after 40 mn, and at 30 L-2-Hydroxyglutaric acid mn intervals thereafter. Following color reaction (should appear reddish), transfer embryos to PBS. == Part 6: Fixation and processing == Transfer to fixation dish comprising PBS, and fix in 4% PFA for 20 mn at.