I recently ended my optogenetics experiment (that I showed the planning for in a previous post), and the next step was to cut the brains. As part of the study, I stimulated the mice 90 mins before perfusion, so that I could immuno stain for c-fos as a marker of neuronal activation. This is important for showing that I can successfully activate my ChR2-expressing neurones, but it also allows me to investigate potential downstream sites of activation throughout the brain. All of which is to say that I had 10 brains to cut, and had to cut and save every section throughout their entirety. Which is a lot of brain sections to cut.
A quick mention of my methods, I am cutting the brains to 30 µm sections on a freezing sledge microtome, and placing the sections in phosphate buffer for free-floating immuno. This is not a technique that I had used, or even seen, before coming to my current lab, so my goal for today is to highlight some of the differences between this method and the one I used previously, namely using a cryostat and doing on-slide immuno.
The microtome is a lovely old-school piece of kit, made of massive cast-iron segments, with a cooling stage to freeze your tissue on, and smooth steel rails to run the stage back and forth on the blade. Speaking of the blade, we use modern microtome blades that fit into a holder, but we still have the original chunk of steel that needs sharpening by hand. For those who’ve never used one, it is slightly odd the first time you use it, because the tissue is frozen on the stage, and when you cut a section it melts at the same time and ends up in a little pile of mush on the blade. You then use a paintbrush to sweep it off and into a well of buffer.
Doing the immuno on floating sections takes some getting used to as well, because you need to suction out the buffer for each step of the immuno, but it takes practice to avoid sucking up the sections (water tension is a bitch). But the most challenging aspect of this method is mounting the sections at the end of the protocol, because you now have to half-float half-push your section onto the slide. It takes a lot of practice to do this in an orderly row, without folding or damaging the sections, and without taking endless hours.
By contrast, mounting sections using a cryostat is easy. Because you do all the cutting and mounting in a frozen chamber, each section is frozen and, for want of a better word, stiff. There are two schools of thought for the mounting procedure: I was originally taught do it onto frozen slides, in which case you manoeuvre the section to where you want it on the slide using a paintbrush then using the warmth of your finger on the underside of the slide you melt the section into place. This works well but it does tend to take a toll on the warmth in your fingers (I manage fine because my circulation is great and my hands are always really warm; the PhD student that taught me had to stop every couple of hours after her fingers became too cold to melt the section into place). The alternative is to leave the slice out on the bench, and then you touch them to the section and they melt on in place.
When you mount sections onto the slides as you cut them, you then have to do the immuno on the slide, which makes washes really easy because you can just dunk them into a well of buffer – no suctioning of tissue to worry about. However, it becomes slightly more challenging to do the antibody steps, because you need to do it on the slide, which means placing the slides flat on the bench and pipetting a few hundred microlitres or so of solution to sit on the slide – in this case water tension is your friend. I found that the bubble of solution on the slide is not level all the way across, which means that you end up with a gradient of antibody and therefore the staining is not uniform across your sections. This doesn’t have to matter much, unless what you need is a quantitative staining.
When it comes to planning which method to use for doing immunohistochemistry, I use the following criteria:
- Use the microtome and free-floating immuno if you want a quantitative readout, such as c-fos, or if you have a lot of brains to cut (I find it’s quicker overall)
- Use the cryostat and on-slide immuno if you are using a method with very low volumes, such as in situ hybridisation, or you want precise placement of sections