In the epidermis. Immunohistochemistry is an efficient means of localizing specific proteins to their expression compartment. In this video, we demonstrate a reliable method for immuno localization of protein in the epidermis, which we use routinely in our laboratory.
Hi, I'm Tammy from Dr.Turin's Laboratory here in the low building of the Ottawa Health Research Institute in the chronic disease program. Today we're gonna show you a procedure for immunohistochemistry using fluorescent antibodies on paraffin embedded tissue sections of mouse epidermis. In our lab, we've optimized protocols for immuno localizing epidermal differentiation markers using BO'S fixed tissue.
As part of optimization, we keep the skin flat by laying it out on paper towel before fixing. This procedure involves dissection of the epidermis fixation, dehydration, paraffin embedding, and section cutting de waxing rehydration antigen unmasking antibody labeling, and finally mounting and visualization with epi fluorescence. So Let's get started.
When using neonatal mice, there's No need to shave the back skin. However, when you have older mice that have fur, you need to shave the back skin away so that it doesn't get in the way of cutting the sections. When you're doing your dissection, you wanna be very careful not to put your forceps in the areas of the skin that you wanna use for your assay.
So what we do is we just put the forceps around the edge of where we're going to cut, and then you lift very carefully, cut the epidermis, remembering not to touch the area that you wanna use for your experiment. When we're fixing the skin, we don't want the skin to roll up and be in the tube. We want it to be nice and flat.
So what we do in order to help that is we lay the skin on a paper towel so that it stays flat while it's being fixed. So now that we have the skin nice and flat on the paper towel, what we do is we cut off the edges or we trim them in order to remove the parts that we may have damaged with the forceps. And then you cut the skin into very small pieces about two millimeter by two millimeter.
Now the tissue is going to be fixed. After cutting the skin into manageable pieces, you immerse it in the boin solution. Cap the tubes, wrap the tubes with perfil and put them on a rocker.
For an overnight incubation, it's important to incubate in the boin solution only 16 to 18 hours. Shorter or longer fixation periods will damage your tissue will. The incubation is over.
You rinse your tissue with PBS and then through a series of ethanol washes using 30%50%and 70%alcohol. You can see that the BO is yellow in color and that as you do the washes, as the boons is diluted out, the color of the wash becomes less and less yellow. You wash two times 30 minutes each time.
Following the last wash in ethanol samples are placed in a fresh 70%ethanol and may be stored at four degrees Celsius until further processing a few vents of paraffin.Embedding. Overusing frozen tissues is that tissue integrity is superior and samples can be stored for long periods of time. In general, core facilities generate more reproducible results.
However, if you have the facility and a dedicated operator who has the expertise, sections can be cut in in-house. Learning to cut good sections is rather an art form. Therefore, we use a core facility.
After receiving the slides back from the Core facility, you can see that the paraffin is present on the slides. As a thin film, the slides are placed on a rack wrapped with aluminum foil, just like a baked potato and incubated for five minutes at 55 degrees. After your incubation is over, quickly unwrap the slides and place the slides in toluene for three washes of five minutes.
It's very important to remember that these five minute washers should be well timed, incubating too long, and toluene will not be good for your specimens. After your three washes and toluene, do a quick rinse in a hundred percent ethanol. Since ethanol and toluene form a precipitate that will interfere with your experiment, this is a very important step to remember.
Next, the samples are rehydrated by incubating slides, five minutes in 95%ethanol, five minutes in, 70%ethanol, then two times five minutes in water. At this point, tissues can be processed for staining with h and e. We also use the core facility for this purpose.
For antigen unmasking poor 500 mils of one time citrate buffer into a one liter plastic beaker and immerse your slides. Remember to remove the metal handle and put your beaker in the microwave. Microwave at high power for two times five minutes.
Remembering to open the door to cool down the microwave in between your two incubations. After your microwaving is done, remove the slides from the boiling citrate buffer and place them into a staining dish containing warm water. We just use warm tap water and the purpose of this is to slowly cool down your slides from boiling to room temperature.
Now you can wash the slides three times five minutes in PBS and we're ready to get going with immunohistochemistry. The first thing we Need to do to begin immuno staining is to dry the area around our specimens with a piece of tissue paper. In order to prevent antibody cross-contamination, we demarcate the area around our specimens with a liquid blocker.
Prepare a humidified chamber by putting a filter paper into a Petri dish. Soak it with water, remove the excess water and insert your slides. The reason why we use a humidified chamber is to assure that during our incubation periods that our slides do not dry up.
It's very important that the slides do not dry because drawing will increase your background. Slides are blocked at this time with incubation buffer For 30 minutes. After blocking, transfer your slides back to the slide rack and incubate in wash buffer.
Remove the excess buffer just as you did before. Put the slide back in the humidified chamber and incubate with primary antibody for one hour. At room temperature.
When your one hour incubation is up, put the slides back in the slide rack and wash with wash buffer for three times 10 minutes. After your washes, remove the excess wash buffer from around your specimen with a tissue paper. Put the slides back in the humidified chamber and incubate for one hour at room temperature with secondary antibody after incubating with secondary antibody, wash in three changes of wash buffer 15 minutes each.
Next, we wash in three times five minutes. In PBS, remove any excess liquids surrounding the specimen and wipe the PBS away from the back of the slide to mount cover slips to the slide. We use mobile with 2.5%DAB co.
In order to reduce fading, add 80 microliters of dropwise to the slides. Being careful not to introduce any bubbles. Then very slowly lower on a cover slip.
Taking care not to leave any bubbles behind. Bubbles are bad because they often land right on your specimen, right where you wanna take your pictures after your mobile is dry. Now you're ready to go Look at your slides.
Here is a look at a successful Staining using an FITC filter. In a 20 x objective, we see Clain six associated with all the super basal layers of the epidermis. Our lab is very interested in the expression and localization of claudins in the epidermis.
With respect to tight junctions and the formation of the epidermal permeability Barrier, we've just shown you how to perform Immunohistochemistry on mouse skin. When doing this procedure, it's important to remember that you want your tissue sections to be flat and that you wanna make sure that your slides don't dry out in between incubations. So that's it.
Thanks for watching and good luck with your experiments.
