Hi, I'm Steve. I work in Dr.Flanagan's lab in the Department of Pathology at uc, Irvine. And today I'm going to show you how to do immunochemistry of human neural precursor cells.
This protocol includes putting cells on lain coated cover slips and blocking and incubating cells in primary antibody overnight, and then doing several was and putting on secondary antibody and at room temperature. And finally, mounting the cover slips onto microscopes flights. Let's get started.
So first I'm going to place three sterile German glass cover slips that are 12 millimeters across into a 24 well plate in order to coat them with laminate. So now I have my cover slips in the plate, and first I'm going to add a solution of PDL poly lysine, and I'm going to incubate the cover subs in PDL for five minutes at room temperature. So now at the end of five minutes, I'm going to remove PDL and put on laminin, and I'm going to incubate the cover slips in laminate for at least four hours or better overnight.
So I've incubated the plate overnight in laminate, and now I'm going to rinse the cover slips with PBS and plate human neural precursor cells that I've already counted. So I just removed the laminate solutions and now I'm going to rein the cover slips with PBS. So now I'm going to remove PBS and put on the media solution.
And finally, I'm ready to see the cells. So I'm going to see the cells at a hundred thousand cells per mill, which means for a 24 well plate I am going to add 50, 000 cells per well, because, because each well contains half a milliliter. So I'm going to tilt the plate from side to side to make sure the cells spread out evenly on the surface of the cover slip.
And now I'm going to put em in the incubator for at least 24 hours to let em settle down and spread out for immunochemistry. So the cells have been growing for 24 hours and I looked at them and they're attached and nicely spread out. So now I'm going to fix them with 4%perfor height for five minutes.
But first I'm going to remove the media and I don't want to let the cells dry out. So I'm going to remove the media and add fixative at the same time. So it's called a two hand technique.
So now I'm going to incubate the cells in fixative for five minutes at room temperature. So it's been five minutes. I'm going to remove PFA and place it in the PFA waste container and add PBS at the same time I remove PFA so that the cells don't dry out.
So I'm gonna use one of these plastic suction pipettes to pull up PFA and I'm going to set the timer for five minutes. So while my cells are in PBS wash, I'm going to prepare a perme visualization solution using Triton X 100. It's a non ionic detergent and it's very, very viscous.
So it's important to wait until all the solution enters and into the pipette. So I'm going to prepare a 0.3%solution in PBS, I'm going to add 10 mils of PBS. And to these 10 mils of PBS, I'm going to add 30 microliters of tri next 100.
So I'm going to add 30 microliters of tri next 100 to 10 mils of PBS. So I'm going to add about 10 seconds to let all all the solution enter the tip. So I, I have here my perme utilization solution.
I put it on the rotator for for 10 minutes, and then I vortex it to make sure the viscous tritton completely dissolved. So now I'm ready to do the perme visualization step. And so I'm going to per wise, for five minutes at room temperature.
So it's been five minutes. I'm going to do now three five minute PBS washes to make sure to wash away all of the triton. So once again, I'm gonna set the clock to five minutes.
So I, I've just finished all of my wash steps, so I'm ready to do the blocking step. So for the blocking step, I've prepared a 5%BSA solution in PBS. And just like with all the washes, I'm going to remove the PBS and add the blocking solution at the same time.
And the blocking step is gonna go for one hour at room temperature. So while my cells are in blocking, I'm going to prepare the plate for primary antibody incubation. So I'm gonna start with this glass plate, and I'm going to cut a, a piece of para film that's about this size.
And what I'm going to do is I'm going to wrap it around and I need some tape, so I'm gonna need four pieces of tape. So I'm just gonna prepare them all. So I'm gonna take this piece of para film and I'm going to straighten it out so that the surface is nice and smooth.
And, and then I'm going to take each end and tape it to the back of the glass plate. So now I'm going to pull this the other end to make sure the surfaces straight, and I'm going to tape the other end to the back. So now I need another piece of pair film the same size as the first one.
And basically I'm going to do same thing. So I taped a second piece of para film on top of the first one, just, just like the first one, except that I left one end of the second piece of perfil loose because I'm going to place the antibody solution and then power slips on the first piece. And then I'm going to place the second piece on top of it that's making a sandwich.
But right now, I'm, I'm going to label the place where I'm going to put the antibody solution and cover. So, so the cells have been in the blocking solution for an hour now, and I'm going to put the cover slips on droplets of primary antibody for overnight incubation now. So I'm going to place a drop of 35 microliters of primary antibody, and I'm going to be very careful not to expel all the liquid because that might create bubbles and bubbles might interfere with binding of the antibody to antigen.
So I want to try to avoid having bubbles in the drop, all three drops of primary antibody, and I'm ready to, to place cover, cover slips with cells onto these drops. So what I'm going to do now is I'm going to pick up a cover slip. So first I'm going to move the cover slip to the side and try to gently lift it with the tweezers open.
As soon as I feel like I've gotten the grip of the cover slip, I'm going to close to slowly close the tweezers and gently lift the cover slip out of the well. So the cells right now are on top of the cover slip. So what I'm going to do is I'm going to invert inverted and gently touch the drop and slowly lay it on top of the drop without creating any bubbles.
And so I'm going to repeat the same procedure with the second and third cover slips. And the final step is to place the second piece of para film on top. And the way I'm going to do it is I'm going to set it down in one motion like that.
I want to try to avoid moving, moving it from side to side. And finally I'm going to secure it to the bottom of the plate with another piece of tape. And now finally, the cells are ready to be incubated at four degrees overnight, and this is the end of day one of immunochemistry.
This is day two of immuno chemistry, and I am going to wash the cover slips in PB S3 times for five minutes. And then I'm going to incubate in secondary antibody for two hours at room temperature. First, I'm going to cut the para film like that so that I can remove the cover slips.
And I'm going to lift the top piece of para film slowly so the the cover slips remain on the bottom piece of care para film. So I've removed the first piece and I have plate with PBS wash solution ready. So I'm going to lift each cover soup and place it in into the well to, to wash off any, any remaining primary antibody that didn't attach to to, to the epitope.
So to lift the cover slip, I'm going to first hold it like that with a razor blade and just gently try to pick it up. And since the cells are, we're facing the antibody solution, I'm going to flip them so that they're on top now and gen the slide it into the well so that there still remain on top. And I'm going to repeat the procedure for the remaining power slips.
And so they're going to be washed for five minutes. So it's been five minutes. I'm going to replaced the PBS solution.
I'm going to add the hooks die. Now I'm going to incubate it for five minutes. So hooks incubation is finished, and now I'm ready to mount cover sos and, and look at them.
So first I'm going to alk quad vector shield, which helps keep the fluorescent dimes from for bleaching. I'm going to, well, first I'm going to label the slide with the, with the numbers of cover slips, and then I'm going to place 10 microliters of vector shield. Vector shield is, is an oil and it's really viscous, so it's, it's also important to to wait a little bit until it all enters into the tip.
So I placed vector shield on the microscope slide, and now I'm ready to place the cover slips on, on the microscope slide. So what I'm going to do first is I'm going to pick up a cover slip and I'm going to rinse the back of the cover slip with deionized water, because if I don't, when it dries out, the salt that was in PBS is gonna crystallize and it's gonna interfere with, with the optical properties of fluorescent dies. So I'm going to, I have my deionized water ready.
And so I'm going to pick up the cover slip and I'm going to rinse the back of it with deionized water. And then I'm going to touch tin wipe to dry it. And then I'm going to invert it again so that the cell side is inverted and goes down onto the microscope slide.
And just like before, I'm going to touch the drop of vector shield and slowly lower it without trapping any bubbles. And I'm going to repeat the same, the same procedure for, for all of the cargo flips. And now let's have a look.
This is an example of what you might get after doing immuno chemistry and obtaining your pictures and making overlays and such. So in this image, this, this is an image of hippocampal neuro stem cells isolated from dent gys in postnatal mice. Today I showed you how to do immuno chemistry of human neural precursor cells.
And if you'll notice, this technique allows you to save the expansive antibody reagent. We used very small volume of primary and secondary antibody by placing a small drop onto a perfil and inverting the cover slip with the cells on top of it. So continue, do doing immunotherapy chemistry, and thank you for watching me.
