This video demonstrates the different steps in whole Mount immunohistochemistry in chick embryo using HRP conjugated secondary antibodies. First, the embryo is fixed in para formaldehyde. Then endogenous peroxidase activity is quenched.
The embryo is then incubated in primary antibody. After several washes, the embryo is incubated in secondary antibody Conjugated to HRP color reaction is revealed using DAB substrate and antibody staining appears orange. 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.
Hi, I am Delphine from the Laboratory of FIN at the Center for Environmental and Genomic Medicine at Texas a and m in Houston. Today we will show you a procedure for performing hormone mount antibodies, stainings in cheek embryos using HRP conjugated antibodies. We use this procedure in the lab to study the morphology of embryos following treatment with antiepileptic drugs.
So let's get started 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 sack with coarse forceps so that the embryo faces upwards.
Next fuse fine scissors to cut a square of yolk sack 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 in yolk and transfer the embryo to a fresh dish containing PBS.
After transferring the embryo, 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 one hour at room temperature to minimize potential microbial contamination. Use D deionized H2O when all of the following steps after the embryo is fixed, aspirate the fixative solution and dispose properly as chemical waste.
Fill the dish with BS.Next, using a microdissection knife, cut a square around the embryo to remove extra embryonic membranes. Remove insect pins with forceps after the pins have been removed. Use a blunt end pasture pipette to transfer the embryo to a scintillation vial containing PBT.
Wash the embryo with PBT three times for 10 minutes each. Remove PBT from the scintillation vial and replaced with PBTX containing 0.3%H 2 0 2 in order to inactivate potential endogenous peroxidase. Incubate for two hours at room temperature on a mutator.
Wash the embryo with PBT first three times for 10 minutes each, then three times for 30 minutes each. To stain the embryo with antibody. Start by washing the embryo for one hour in blocking buffer or 1%BSA 1%NGS.
We use NGS because the secondary antibody is raised in goat. Incubate on mutator for one hour at room temperature after incubation, dilute the primary antibody one-to-one in blocking buffer and incubate embryo in this solution for two days at four degrees Celsius. On mutator.
The primary antibody dilution factor depends on the choice of primary antibody. Here we use an antibody from the developmental studies hybrid DOMA bank, which is provided as super natin. We dilute it one-to-one in blocking buffer.
Next, perform three 10 minute washes in PBT followed by three one hour washes in PBT. After washing, dilute the secondary antibody one to 2, 500 in blocking buffer. In this case peroxidase conjugated goat anti-US IgG and incubate embryo in this solution overnight at four degrees Celsius on mutator.
Perform three 10 minute washes in PBT followed by three one hour washes in PBT to develop the color reaction of the stained embryo. First two 20 minute washes in tris buffer 100 millimolar tris, HCL pH 7.4. Next, remove the last TS buffer wash from the vial containing the embryo and replace it with five milliliters DAB solution.
Keep it in the dark on the mutator for 20 minutes. Dispose of the einor tip in the bucket containing a 10%bleach solution. In order to decontaminate DAB, add 16 microliters.
Stock H 2 0 2 to the vial containing embryos in DAB. Keep it in the dark After 10 minutes, monitor the reaction under microscope when the color reaction is complete. Dispose of DAB in bleach bucket, replace with five milliliters.
Tap water. Next, perform two 10 minute washes in PBS to process for photography and wax sectioning. Dehydrate the stained embryo in an ethanol series, 25%50%75%and 100%for 10 minutes each.
After dehydration, replace the ethanol with 0.5 to one milliliter cedarwood oil. This will make the embryo translucent process for photography in cedarwood oil. Following photography, return the embryo to the vial and replace the cedarwood oil with 100%ethanol.
Finally, replace ethanol with 5%fast green FCF in 100%ethanol and incubate for three minutes. This step will make embryos visible for histology. Replace the fast green FCF solution with 100%ethanol and process for histology.
Here are three examples of stained embryos. These stage 11 embryos are labeled with 15.3 B nine or not. One a nodal cord specific antibody developed in the laboratory of Jane dod and Tom Gessel.
After two minutes in DAB, the embryo is still transparent. After 20 minutes in DAB, slight labeling appears in the nodal cord. Finally, after 40 minutes, labeling of the nodal cord is complete.
In this example, we show how PAC seven an antibody developed in Tom Jessel's laboratory recognizes early stages of neural tube closure as well as the cranial neural crest cells emerging from the midbrain. Here the embryo is labeled with Croc 20 probe from the laboratory of David Wilkinson, followed by not one antibody. We've just shown you how to perform whole mount immunohistochemistry uncheck embryos.
This procedure can also be used on embryos which have been previously processed for whole mount in situ hybridization. In this case, briefly fix the embryo for one hour following in-situ hybridization and then proceed with antibody staining. So that's it.
Thanks for watching and good luck with your experiments.
