Following fixation for 18-24h I store the whole brains in PBS containing Na-azide at 4C. On the sectioning day, I placed the brain on the freezing stage using couple drops of OCT applied to the base. Then I gradually put additional OCT to cover it. I start sectioning when the OCT turns into white and set the temperature to -18 -20. My main problem is the sections are rolling and impaired. Thanks in advance.
First, fixation is the most critical step affecting slicing in the cryostat, vibrating microtome, and microtome—whole brain immersion in fixative results in poor preservation. Autolysis occurs in deeper brain structures, and slicing will be almost impossible. Even perfusion fixation without proper care may impact slicing. Remember that slicing a rat/mouse brain is usually a challenge because we have many structures with different densities lining each other, one of the reasons that makes it difficult to slice.
If the fixation is okay, you must soak the brains in cryoprotectant solution (2.3M sucrose). Change the solution every 6 hours for one day or two. Then, you can freeze the brain. I recommend you embed the brain in Tissue Freezing Medium with a plastic mold. By doing so, you will get a solid bloc. It will facilitate slicing. If wrinkling persists, you can also place the slice in a buffer-filled jar and then recover the slice in a glass. The freezing mounting media will dissolve in the buffer, and the section can be easily placed onto a glass slide.
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