The "Cryostat" is a device that keeps your sample or devices at a stable low temperature, we use a cryostat microtome or simply a cryostat to slice brains into thin sections for staining to check placement of cannula, tissues, viral injections etc. In addition we have microtome in the lab, that can be used instead.
The Cryostat is located in MSBII on first floor in a secure area you will require card access to enter the area this can be done by contacting Kristie Flanders kmflande@indiana.edu and request access, you also need to go to her office on the ground floor of MSBII and sign a form.
Important: The cryostat is a shared piece of equipment this means that you must sign up to use it using google calendar ( the cryostat calendar can be found on the newmanmorylab gmail calendar), and that you must clean up the area when you are done using it. Further you must be trained to use the cryostat properly before you use for the first time. Reading this protocol is not sufficient for using the cryostat. Ask around the lab and have someone who knows how to use it teach you.
Materials ( Most of items are kept in the histology box)
Tissue tek
The brains you are going to slice
Slides
Glass plate
gloves
slide box
Razor blade regular
Razor blade thin
Rat or Mouse Atlas (for beginners)
Chucks
Graphite pencil
Cryostat operation
First check the temperature of the cyrostat it should be to slice between -18 and -22 degrees in addition hit the light bulb light to turn the light on.
The knob in the center with small numbers is how you control the thickness of the slices we keep are slices normally at 40um.
1. First take chuck or chucks out and place a small amount of tissue tek on the top and place them in the cryostat and place the small piston over them to create a smooth surface to place the brains on.
2. After the tissue check is completely white, knock off the piston and take the brain you are going to slice out of the jar and slice through the cerebellum or between the cerebellum and the cortex to create a flat surface.
3. place some tissue tek on top of the chuck again and quickly place the brain onto the chuck and make sure it is centered, before place a thin layer of tissue tek around the brain.
4. Place the chuck with brain back in the cryostat. It will take 10-15mins for the brain to get down to temperature to slice it.
5. Replace the small glass plate in the cyrostat if it is in there for our own. Important: Do not forget to take our glass plate out after you are done slicing.
6. Place a thin razor blade into the holder and lock it into place. Take your slides that you have and write on the bottom of each one using a graphite pencil the name of the animal your name or initials, the date and C or F followed by a number C for cresyl violet staining F for Florescence staining number to keep track of which slide is which, so start with C1 through Cn to you have collected all your tissue.
7. Once your brains are frozen, and you can tell by getting clean slices if your slices tend to mush together the brain is not frozen enough the brain should when frozen be rather pale.
8. Place the chuck into the hold and turn the screw to make it tight, again make sure to center the brain.
9. Using the direction buttons slowly advance the brain forward so that you just begin to get slices from the brain. When using the spin wheel to move the brain past the razor blade do not spin it too fast or too slow, otherwise you can damage the wheel if you spin to fast and affect the quality of your slices.
10. The most difficult part of using the cryostat is making sure the glass plate is in the proper distance from the razor blade if the glass plate is to far back it will not catch the brain slices, if it too far forward the brain will just bump into the glass plate and you won't get any slice at all. To adjust it make small adjustments to the black knob to advance or pull back the glass plate. This is a little bit of trial and error again make small movements otherwise it will take you forever to adjust it to the correct position. Chances are it is only off a little bit if you are not getting clean slices, so large adjustments are not necessary.
11. Important: Be certain that before you reach your area of interest you can collect nice clean slices. When you are lifting the glass plate up and down off the metal plate do not slam it, this will cause tiny cracks in the glass affecting the quality of the slices.
12. When slicing I recommend to save time you slice the number of slices that you can fit on one slide at a time which differs depending on what area of the brain you are in.
13. After you have the slices carefully list up the glass plate, the slices tend to stick to the edge of the glass plate and need to be pushed off using one of the brushes in the cyrostat Important: Remember that the slice will stick to anything that is warm.
14. Using the slide carefully pick up each of the slices. When you are finished place all the slides into the appropriate slide box. and clean up the cryostat.
Clean Up:
Important: Do not use water to clean the cryostat, the cryostat is very cold if you use water you will create ice in the cryostat. In the cryostat room there should be clearly labeled bottles of 70% ethanol use this to clean the cryostat.
Do not leave the razor blade clamped into place remove it after use.
Remove our glass plate and wipe down gently with ethanol
Wipe down the metal plate with ethanol
Remove the tissue collection tray and dump all the uncollected slices into the trash
Turn off the lightbulb in the cryostat before you leave
Make sure you do not leave anything behind
Discard what is left of the brain into the trash or keep for practice histology but be sure to label glass jar as such.