Dear Dmitry,

This article has some interesting information

Hi Dmitry,

I do it in two different ways depending on the experiment I'm running.

1) I perfuse the tissue with PBS followed by 4% PFA, postfix it in 4% PFA at room temperature for 4h and then I rinse it with PBS 3 times and leave it in 10-30% sucrose solution shaking at 4 celsius degrees until the tissue has sunk in the sucrose. Then I freeze it in dry ice and leave it at -80 C until sectioning.

2) Snap freeze the tissue fresh with cold isopentane (about -30 to -35 degrees) and leave it at -80 until sectioning.

Nr 1 I usually do for immunohistochemistry and nr 2 for in situ hybridization. Maybe you could try both and see what works best. Good luck.

Dear Dmitry,

I’ve been using lab-made cryomolds (made out of basic aluminum foil from the general grocery store) and Tissue Tek O.C.T. tissue-immersion before snap freezing in liquid nitrogen for skin, liver, spleen and lymph nodes and after some initial adjustments, I’ve had no problem thus far.

This is what I do:

After obtaining the tissue of interest, I fill half of the cryomold with Tissue Tek O.C.T. Then, I add the tissue sample to the mold and fill the other half until the sample is completely submerged. After the tissue is completely submerged I wait 15 minutes (at room temperature) and then snap freeze it in liquid nitrogen. Finally, store at -80°C.

I’ve used the frozen tissue sections for general histopathology staining and immunohistochemistry with great results.

I hope any of this information is useful for you.

-Angel

Freeze a fresh, unfixed tissue sample, up to 2.0 cm in diameter, in OCT in a suitable tissue mold. Freeze the OCT containing the tissue onto the specialized metal grids that fit onto the cryostat.

OCT is viscous at room temperature and miscible with H2O, but freezes into a solid support at −20°C.

I have used fresh frozen sections of gonads for localization of steroidogenic enzymes and lipids. Fresh tissue taken on a tissue holder was directly frozen using carbon dioxide and fixed to cryostat to take sections of 14 micron. Sections were taken on a cover glass and thawed prior to exposing to enzyme substrates and placing for incubation. Glycerol was used as mountant. I have Not used any cryoprotectant or preservative.