[Histonet] isopentane freezing of rat brain

[Hector Gonzalez Pardo]

Hi,
I am using for the first time isopentane (2-methylbutane) to freeze my rat brains for histochemistry. However, when I do the histochemical staining, the tissue looks like "granulated" under the microscope, especially in the center. We usually immerse slowly (30 secs.) the entire brain on a weighing plastic boat in a glass vase containing isopentane chilled at -40 ºC. Then we wait for another 30 secs until it looks white on the surface, and we store it at -40ºC until sectioning using a cryostate. We used before a time-consuming procedure that worked, using cryoprotection with sucrose overnight, covering the whole brain with a cryogel (OCT compound / Tissue-Tek) and using a kind of freon-22 cooled by liquid nitrogen. Since we would like to continue using the easier isopentane procedure, does anyone know what are we doing wrong? Thanks!