This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1. Open in a separate window Click here to view.(64M, flv) Protocol I. Schematic Overview: This Regorafenib Hydrochloride video demonstrates the different steps in whole mount immunohistochemistry in chick embryo. First, the embryo is fixed in PFA [IHC1]. Regorafenib Hydrochloride Then, endogenous peroxidase activity is quenched [IHC2]. The embryo is then incubated in primary antibody [IHC3]. After several washes, the embryo is incubated in secondary antibody [IHC4]; Color reaction is revealed using DAB [IHC5] and antibody staining appears orange [IHC6]. Part 1: Fixing the embryos To perform whole mount immunohistochemistry on chick embryos, first open an egg by tapping the shell with forceps and removing pieces of the shell. Remove the thick albumin with forceps, and tilt the yolk sac with coarse forceps so that the embryo faces upwards. Using fine scissors, cut a square of yolk sac around the embryo. Remove the embryo from the yolk with a spoon, and place in a dish containing PBS. Under a dissection microscope, carefully remove the membranes and yolk and the embryo Regorafenib Hydrochloride transfer to a fresh dish containing PBS. Pin the embryo down with forceps and insect pins, and aspirate the PBS. Replace this with 4% PFA in PBS, and allow the embryo to fix 1h at RT. Part 2: Preparing embryos for antibody step To minimize potential microbial contamination, use ddH2O in all following steps. After the embryo is fixed, aspirate the fixative solution and dispose properly as chemical waste. Fill the dish with PBS. next, Regorafenib Hydrochloride using a microdissection knife, cut a square around embryo to remove extraembryonic membranes. Remove insect pins with forceps. After the pins have been removed, use a blunt end Pasteur pipette to transfer the embryo to a scintillation vial containing with PBT (PBS pH 7.4, 0.5% Triton X). Wash the embryo with PBT, 3 times for 10 minutes each. Remove PBT from the scintillation vial, and replace with PBTx containing 0.3% H2O2 in order to inactivate potential endogenous peroxidase. Incubate for 2 hr at RT on nutator. Wash embryo with PBT, 3 times for 10 minutes each, then 3 times for 30 minutes each. Part 3: Antibody incubation To stain the embryo with antibody, start by washing the embryo for one hour in blocking buffer or 1% BSA/1% NGS/PBT (we use NGS because the secondary antibody is raised in goat). Incubate on nutator for one hour at RT. Dilute the primary antibody 1:1 in blocking buffer, and incubate embryo in this solution for 2 days at 4C on nutator. Primary antibody dilution factor depends on the choice of primary antibody. Here, we use an antibody from the Developmental Studies Hybridoma Bank, which is provided as supernatent . We dilute it 1:1 in blocking buffer. next, perform 3 10-minute washes in PBT, followed by 3 1-hour washes in PBT. After washing, dilute the secondary antibody Rabbit polyclonal to Caspase 7 1:2500 in blocking buffer (in this case peroxidase conjugated-goat anti-mouse IgG (H+L), and incubate embryo in this solution O/N at 4C on nutator. Perform 3 10-minute washes in PBT, followed by 3 1-hour washes in PBT. Part 3: Color reaction To develop the color reaction of the stained embryo, first perform 2 20-minute washes in Tris buffer (100mM Tris HCl, pH 7.4). Meanwhile dissolve DAB substrate (3,3-diaminobenzidine tetrahydrochloride) in Tris buffer at 500g/ml under fume hood; keep solution in dark, on ice. Remove last Tris buffer wash from the vial containing embryo and replace with 5ml DAB solution from step 3 3.2. Keep in dark on nutator for 20 mn. Dispose of Eppendorf tip in bucket containing a 10% bleach solution in order to decontaminate DAB..