How to use a cryostat

1)Bring your cryomoldondry ice, also bring black slide box to keep your cut slides

2)The machine is always on (cold at -20C)

3)Turn on the light (light bulb button on the top control panel)

4)Use OCT to mount your samples (bottom of the mold – up, so that the samples will face the blade right away)

5)Mount your samples onto the holder, make sure it’s tightly secured

6)Adjust the distance between the sample and the blade: usually bring the sample to the blade using orbutton on the left panel

7)Trim the mold using “TRIM” button until you see the sample

8)**It’s important to “LOCK” the stage when you are not cutting – right hand side switch** when it’s lock the display will say “LOCKED”

9)Cancel the trim by pushing trim button again (this will still trim one more time before it’s canceled)

10)Select the thickness of the sections using + or – button on the left panel: usually 10-12 μm for female reproductive tract.

11)Cut the tissues few more times, the very first times, the sections will still wrinkle, cut few more time continuously, the sections will be less folded.

12)Don’t forget to use anti-roll guard so that the tissues will not roll.

13)Try to see if the section has the desired tissue, put 1 section on charged slide (make sure you’re using the right side of the slide)

  1. To do this you use warm slide to pick up the section
  2. Prior to that, you will use the small paint brushes (2 of them) to separate the sections (after you cut the ribbon of tissues).
  3. Paint brushes work better than forceps.
  4. Touch the section very gently, so that the tissue will not break
  5. After that use the warm slide to pick up the section, the OCT will melt and look translucent
  6. Alternatively, you can pre-chill the slide inside the cryostat and pick up the section with the chilled slide – it’s whatever you prefer.

14)Look under light microscope whether it’s the tissue you want

15)If you see the tissues you want, cut some more and put them on charged slides, labeled with pencil.

16)**MAKE SURE YOU CLEAN UP THE CRYOSTAT**

17)Use 70%EtOH to wipe the OCT smudge or sections inside the cryostat. If the tray is needed to be washed, you can wash them and wipe dry before you put it back in the cryostat

18)Keep the slides in dry ice then transfer to -20C or -80C until stain

19)You can mount cryosection with ProlongGold with DAPI and sealed with nail polish at room temperature overnight. The tissues can stay at room temp for few days.

20)If you want to keep long term – the fluorescence will last for few weeks. So, if you are not stain right away, keep the cut sections at -80C.

21)Image the sections right away after staining to prevent the fading of the fluorescence.

Winuthayanon Lab