I am trying to section rat brains for IHC. Typically, we perfuse transcardially with 4% PFA, post-fix in 4% PFA, and store in 30% sucrose before sectioning on a cryostat. I am trying to label a protein that is ~125kD and expressed throughout the cell in both soma and neurites and look for coexpression in a particular cell type.
I have attempted section widths between 10um and 25um, direct to mount. However, even with many tricks (e.g. keeping slides in cryostat and slowly warming for the tissue to adhere), I am not satisfied with the sections. That is, there are bubbles or folds in the tissue formed as it melts and adheres to the slide. I have previously worked with free-floating sections for other protocols, but am concerned that using free-floating sections will require thicker sections, and that may compromise our ability to accurately identify (or rule out) coexpression.
Embedding your tissue in gelatin or OCT before embedding may help. Let your blocks equalize to temperature in the cryostat, for at least 30 min before you start sectioning. It also helps to let them dry for about 2 hours after you section, before you start any procedures. If you are getting folds, the problem may be that as you are sectioning, the tissue is wrinkling. If you do gelatin embedding you may try to guide and straighten your tissue with a tiny paint brush before you mount on the slide, so that the section is neat and straight.
I'll echo the other writers and say that embedding in OCT is a good idea as well as using a fresh blade when you start. I always cut very thin slices for my studies (around 14 microns) and would use room temp superfrost plus slides to pick and instantly adhere the tissue. It takes practice and steady hands, but you can learn to pick up slices with little or no folding. Temperature control in the cryostat is essential though. OCT that is too warm will start to melt, but OCT that is too cold will produce slices with nicks and folds where the blade got briefly caught by the OCT or tissue.
Thanks, all. I do use OCT. What I find is that the OCT seems to melt to the slide more quickly than the tissue, pulling the tissue and creating the bubbles. I have tried adjusting the amount of OCT to try minimizing the curling of the tissue as it comes off the blade. The best I've been able to manage is mounting the brain upside down (since my AOI is very ventral) and use paintbrushes to try to flatten my sections as best as possible on the cold stage before rolling the slides atop to the tissue and using my finger to slowly warm the tissue to the slide.
Free-floating sections of PFA-fixed rodent brain can be freeze-cut (preferably from 20% glycerol/2% DMSO/PBS rather than sucrose) as thin as 10 microns, although 15 micron sections are easier to handle; sections are then collected into cryoprotectant, e.g. 50% ethylene glycol/0.1%PVP/PBS, and stored at -20C. Sections can be mounted on gelatin-coated slides from a medium containing protein stabilizer and volatile buffer; for this we currently use 5% trehalose in 0.1M triethylamine acetate pH 7.0 Alternatively, at >/= 15 microns, sections can be stained under free-floating conditions. The only advantage of mounted sections is for co-localization using Proximity Ligation Assays when the co-localization is at the molecular level.
You may also try this: Cut the block until a small part at the upper end of the block, in that way, that the section is still adhering to block. Then turn the wheel back, so you see the whole block and the section again. Use the brush to flatten the section onto the block's surface. Then take a roomtemp. glass-slide and move closely to the lower part of the block/section, in that way, that the section just sticks on the glass with the lower edge. Then you must try to pull and mount the section at the same time, without loosing the connection to the block. The pulling should flatten the section.
In my experience using a cold slide and warming it afterwards brings more folds than wanted.
You can cut your section in the cryostat 10um -25um and with the brush drop the section in the water. It is floating and smooth. Fish your section, grab the section with the superfrost plus slide, stand up and leave it dry. In 15-20 minutes put in PBS and start the IHC staining or put in mailer box and store it at -20 or -80 until to use. Guaranteed your section will be smooth, no bubbles or folds.
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