Setting Up the Cryostat

• Brushes

• Sample Holders ( chucks ) :

  • Freeze the tissue onto the metal disk
  • Its what actually holds the sample in place while sectioning occurs

  

• Put into box to "pre-chill"

  

• Cryostat is kept at $-{20}^{\circ }C$$-20^{\circ}\ C$

  

• Sectioning Device

  • Every time it goes through a cycle , it will advance the section holder a little bit to the front

  

• Single Use Cryostat Blades

  • 1 Edge Razor Blades

  

• Optimal Cutting Temperature Compound ( O.C.T. )

  • Used to mount tissue onto the chuck
  • Starts out as clear liquid , but when it freezes , it turns opaque = "dark , shaded , unlit" = "impervious to the rays of light"

  

• Put on top of tissue sample , which is on top of chuck :

  

• Turns white as it freezes :

  

• Slides :

  

• Roll Guard :

  • prevents the tissue samples from "rolling up" as you are cutting them
  • take in and out every time you use the cyrostat :

  

Setup Sequence

1. Check positioning of sample holder

   • should be set to around ${11}^{\circ }$$11 ^{\circ}$

   • each mark = $2^{\circ }$$2 ^{\circ}$

     

2. Blade Holder Lock - Forward / Back

   • You want to bring the whole apparatus close to where the tissue is on the sectioning device

     

3. Orange Lock = allows for pivoting of sectioning device

   • Normally , you don't need to mess with this

     

4. Open "plate" so you can insert the Roll Guard ( glass piece )

   • Make sure grooves are facing down

     • so that as the tissue comes underneath the roll plate , it has clearance space

   • this step should be done early in the whole setup sequence , because the roll plate starts at room temperature , and needs time to cool down

     

     

5. Verify tissue sample is frozen ( O.C.T should be solid white )

   

6. Mount tissue sample into sectioning device :

   

7. Tighten Pivot Knob to keep sample from rotating

   

8. Get a blade out of the blade dispenser

   • Pull black slider piece with arrow all the way back

   • Then push black arrow piece slowly forward , should expose a single blade

     

9. Attach blade to cryostat

   • there is a tiny groove that it drops into

   • make sure it is centered ( left and right )

   • make sure it is not off at a weird angle ( should be flush with roll guard plate )

     

10. Use right-side orange locking handle to clamp the blade into place