Cryostat Brain Slicing

Purpose:    The following steps will prepare mounted slices of fixed brain from frozen tissue.   Slicing a frozen brain will allow sections as thin as a few μm.   These preparations can be used in  staining   and in  immunohistochemistry .

Materials

       Fixed, dehydrated rat brain

       5 mL plastic beaker to use as a mold

Embedding medium for freezing

Cryostat [Microm Vacutome HM 500 OM]

Coated slides

Slide warmer  

1)       Place the fixed, dehydrated brain in a small mold, such as a 5 mL disposable beaker.   Fill beaker with embedding medium and freeze at -800 C until solid.

2)       Check that the cryostat is turned on and operational.

3)       Warm several slides in an operating slide warmer, frosted side up.   This will prepare them to mount the frozen slices.   (Instant thawing of slices onto the warm slides will help them adhere to the coating.)

4)       Set the cryostat to Trim/Feed.   Set Trim to xx (for xx μm slices) and Feed to 90.

5)       Slightly thaw the mold holding the frozen brain.   Remove the frozen block and place on the cryostat stage.   Select “Freeze Object”.   The chamber temperature will drop to freezing.

6)       Adjust the orientation brain/block for optimal slicing.   The cryostat allows rotations of 5o   in all directions.

7)       Adjust the blade orientation to be square with the brain/block.

8)       Feed the block forward 90 μm by cranking the manual dial.   Make the first slice.

9)       Switch to Trim mode before making additional slices.   Manually advance the blade before each slice.   Keep the blade clean between slices by brushing away debris.   Check slices for hippocampal tissue.   Slices without hippocampus can be discarded.

10)   When the hippocampus becomes visible:

a.        Lift the blade and stage

b.       Press a warm slide onto the slice, frosted side down.   Note: If the slide is not warm, rub the back with your fingers.

c.        Each slide can hold multiples slices.   Take care that they do not overlap.

Note:    Temperature inside the cryostat chamber will vary with external temperature.   Slices will tend to curl if they are thawing too quickly.   If curling is evident, adjust the temperature.  

11)   When slicing is complete, clear the cryostat chamber of debris.

12)   Label and freeze slides at -80o C until needed.

Operational Note:    When the vacutome is in “Sectioning mode”, the blade will advance automatically slices without manual cranking.

From  the lab of Lucien T. "Tres" Thompson, Ph.D. The University of Texas at Dallas