Frozen Section protocol for cryo UltraMicrotome.
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Frozen section protocol
Day 1:
Add 3 ml of freshly made fix to vial and put vial on ice.
Dissect at least 10 eyes trying to get as little head as possible left on the eye.
Put in fix and incubate on rotator in the cold room for 1 hour.
Remove fix and rinse 1 X with cold PBS.
Add 3 ml cold 40% sucrose to vial and incubate on rotator in cold room over night.
Make sure there is enough liquid nitrogen, if not order now. You should ask for a 50 psi working pressure tank.
Day 2:
Fill tank:
Attach filler tube to liquid port on the small and large tank.
Turn on the valve on the large tank, open the vent valve on the small tank, open the liquid valve on the small tank. This is a very noisy process int he beginning.
Monitor the pressure gauge on the small tank. If the pressure begins to exceed 15 psi, partially close the liquid valve on the small tank. As the tank fills and cools the pressure should drop and allow you to open the liquid valve all the way.
The small tank is full when liquid nitrogen starts coming out of the vent.
Close the liquid valve on the small tank and then close the vent valve on the small tank.
Finally close the liquid valve on the large tank.
Detach the liquid nitrogen filling hose from the small tank. Care should be taken because sometimes there is liquid nitrogen remaining in the hose, so be sure to wear the cryo-gloves.
Insert knives into the microtome:
Put knives in the knife holder.
The knives should be pushed all the way forward so that the fronts of the knives are touching the cross bar.
Try to select knives of similar size if you plan on cutting more than 3-4 samples.
Use the largest allen wrench to tighten the knives with the set screw on the side of the knife holder.
Put knife holder in the microtome being careful not to hit the blades with anything.
Tighten the knife holder using the second to smallest allen wrench. The set screw is on the right side on top of the knife holder mount closest to you.
The set screw further back adjusts the knife angle and should not be touched.
Put Plexiglass cover on top of the cryo-chamber.
Cool microtome to cutting temperature:
Mount filler hose on the bench with the chamber end sticking through the hole provided in the Plexiglass. The hose shouldn't touch the sides of the plexiglass. Put dish under hose. Make sure all doors on the Plexiglass are closed.
Attach the filler hose to the small tank via the mt-5000 port. Use a wrench to tighten the nut. Pliers will strip the ut and it is a pain to replace.
Attach the temperature control unit to the tank using the serial cable.
Turn on temperature control unit, and set sample temperature to -230 and the knife temperature to -500.
Open mt-5000 valve on the tank, and press nitrogen filling button on temperature control unit.
Turn on microtome and reset the cutting arm to the fully retracted position.
It will take about a half an hour for the unit to fill with liquid and the temperature to stabilize.
Mount eyes on aluminum stubs:
Using a Pasture pipette, suck up a little sucrose from the vial, and then suck up five eyes as closely packed together as possible. You can get your eyes in two or three steps but it is more of a pain.
Deposit the eyes on the stub. At this point you will have much more sucrose than needed on the stub. To suck some of the sucrose off, position the pipette on the edge of the stub with only a little bit of the pipette sticking over the top. Suck off sucrose until the eyes are just covered but not floating.
Using a tip of the green forceps arrange the eyes in a cluster in the center of the stub making sure that all of the eyes are positioned lens side up. Deposit stub into pool of liquid nitrogen in the cryo-chamber.
After the liquid nitrogen stops boiling use the forceps to pick up the stub. You will want the end with the eyes to be facing you. Inser the stub into the sample holder and push as far back as it will go. Tighten the stub using the largest allen wrench.
Cutting sections:
Start on one side of the knife. The knife can be moved side-to-side using the knob on the right side of the stage.
Using the knife positioning knob (on left of stage), pull knives toward you so that when the sample is lowered it will not hit the knives.
Using a rocking motion with the sample position wheel raise and lower the sample so that it goes above and below the knife edge. Each time you raise the sample move the knives forward a small amount. You want the first contact between the sample and the knife to shave off only a very small amount of the sample.
Using the rocking technique shave off our or five more sections. At this point you can turn the microtome onto automatic mode or continue manually by cranking the sample position wheel. Remember to only crank clockwise (your hand is moving away from you at the top of the stroke), because it is possible to damage the microtome by going through one full revolution counter-clockwise.
Use eyelash on a stick to gently move sections away from the knife edge, and continue collecting sections until you have about 10.
If the sections curl or smush the knife temp is too warm. If the sections crumble the knife temp is too cold.
Dip loop in 23 M sucrose. The drop of sucrose solution on the loop should be fairly flat if it is too big the sections will spread out on the surface.
Lower the loop so that it is almost touching the sections that you want to pick up. The sections should jump up onto the sucrose.
Be careful not to touch the sucrose to the knife or it will stick and cause a big mess on the knife and ruin any sections in the sucrose.
Gently put drop of sucrose on treated slide, in a humidified chamber. Continue collecting sections until you have four to five drops on each slide.
Stain slides:
Rinse sucrose off the slides using PBS-T in slide washer.
You will need 50 µl of diluted antibody for each slide. Mix 1 µl of antibody with 100 µl with PBS- BSA for every 2 slides you are going to probe.
Pull slide out of PBS-T and use a Kimwipe to remove all of the excess PBS-T except for the circle where the sections are. You want to remove any standing buffer from the circle but do not dry it out.
Add 50 µl of the diluted antibody to the circle and return the slide to the humidified chamber.
Incubate the slide at 4° overnight.
Wash 3 x 15 minutes in PBS-T.
Repeat above for secondary except incubate 4 hours at room temperature in the dark.
Wash 3 x 15 minutes in PBS-T
Remove PBS-T from the slide taking care to remove standing PBS-T from the circle without drying it out.
Add 15 µl Prolong antifade reagent and cover with cover slip.
Let the slide stand in the dark for at least an hour and preferably over night before looking at it.
Solutions:
Fix
4% paraformaldehyde in PBS
Heat a 500 ml beaker of water to boiling in microwave
In a 15 ml conical tube add .4 g paraformaldehyde to 9.8 ml PBS
Vortex
Float tube in hot water vortexing every 2-3 minutes until dissolved.
Cool in ice
40% Sucrose in PBS
Add 40 g sucrose to 50 ml PS and dissolve.
Bring up to 100 ml with PBS.
Store at 4°C.
2.3 M sucrose in PBS
78.7 sucrose in 30 ml PBS and dissovle
Bring up to 100 ml with PBS
Store at 4°C.
Block
4% BSA in PBS-T
Store at 4°C.
1. Sample Preparation
A. Dissect eyes into 4% formaldehyde (1xPBS) on ice, using small cylindrical vials
B. Fix tissue for 1hr, 4°C, shaking
C. Wash 1x in cold 1xPBS
D. Infuse/maintain in 40% sucrose/PBS, 4°C, O/N
II. Slide Preparation – Silicon/Wash/Gelatin Slide Preparation for Frozen Sections
A. SigmaCote (c) Slides:
1. Place ~80mL of SigmaCote (-20°) in a 100mL beaker inside hood
2. Place ~10-15 frosted slides into beaker, wetting sides and avoiding frosted area
3. After wetting, place slides into box to dry under hood for ~30 minutes
when drying, orient all slides frosted-side facign forward in box
B. Clearing site for gelatin with 50% NaOH (in water)
1. Arrange dried slides frosted side up on bench
2. Using a P100, place a sufficient amount of 50% NaOH on the center of each slide, forming a circle
3. Allow slides to sit for a minimum of 1.5 hours (~2 hours is sufficient; more than 2 hours dries the NaOH making it more difficult to remove)
C. Remove NaOH and Wash slides
1. Suck off NaOH circles using sink-supplied vacuum
rinse hose with water several times to prevent degradation from NaOH
2. Place slides in black tray/fly tray or basin with holes (one layer) under running wateroccasionally rotate slides by hand and exchange for fresh water
D. Gelatinize Slides - "SUB" solution ( see recipe in Drosophila Protocol book, or below)
1. Dry slides by gently absorbing moisture with Kim Wipes (large)
2. Dip slides into beaker or Sub solution and set to dry flat on bench
3. Once dry, store O/N in slide box
keep box open O/N on bench to facilitate drying
4. Recycle remaining Sub solution - return to main bottle
"SUB" solution recipe: p. 231, Drosophila Protocols
a) Dissolve 0.5g gelatin and 0.5g chromium potassium sulfate in 500ml of hot water (just boiled is fine)
b) Stir on a hot plate until dissolved (~2 hours)
c) Store at room tempif made previously, microwave to reach close boil to sterilize
SUB solution cannot be filter sterilized
III. Protocol for Preparing Microtome/Liquid N2 for Frozen Sections
A. Fill small tank (do not let pressure exceed '20") from large tank
B. Small tank will take about 10 minutes to cool, 10-15 min. to fill
C. Using small tank with Microtome:
1. Connect small tank to foam-enclosed tube that fits in microtome reservoir
2. Connect red box to small tank with parallel/axial cable
turn red box "ON"
4. On the red box, set specimen temp to -20°C and set knife temp to -50°C
5. Turn on ("in/down") nitrogen flow button
6. Open small tank liquid valve - do NOT open vent once tank is filled
7. Wait about 20 minutes before using
D. Sample preparation for slicing:samples should be fixed in formaldehyde (4%) and contained in sucrose (40%) in PBS
1. Using a Pasteur pipette and hand-controlled pipetter, suck up 4-5 eyes and sucrose
2. Place sucrose + eyes on etched surface of stub under small dissecting scope
3. Orient eyes lens up with forceps and adjust sucrose amount to cover eyes sufficiently - do not let eyes float
4. using forceps, hold prepared stub in dish of liquid nitrogen for 5-10 seconds and then let it drop in (note orientation of stub for next step)
5. Pick up stub and insert it into microtome sample holder quickly, but carefully, sample facing knives
6. Tighten stub in microtome
7. Allow microtome to reach sample temperature before continuing
E. Frozen sectioning:
1. Use eyelash to slide slides down knife as additional slices are being cut
2. When microtome beeps 7 times, microtome arm will automatically retract; slicing will stop
F. Placing samples on slides:
1. Dip loop into 3.2M sucrose with no excess/no drip
2. Use sucrose in loop to life slices off of knife and to place onto slidesnote: take care not to freeze loop to knife (do NOT allow loop to touch anything other than slice)
3. When all slices have been lifted and placed on slide, continue slicing
4. Keep all slides hydrated during and after use in large Petri dish with wet tissue lining
G. Cleaning up and Shutting system down:
1. Wash stubs by holding with forceps under running water; shake dry
2. Wash loop by holding under lightly running water; carefully remove all sucrose
3. Turn off microtome
4. Turn off nitrogen level button on red box
5. Disconnect axial cable between red box and small tank
6. Turn off "microtome MT5000" small tank valve and place tube aside to dry
note: do NOT vent tank - all remaining liquid nitrogen left will be lost
7. Raise both sample and knife temperature on red box to +30° to help warm microtome
8. Remove dish and pour liquid nitrogen out onto the floor; place dish on bench to warm
9. Remove microtome plastic cover and place in sink
10. Unscrew knife receptacle and remove from microtome; let warm
IV. Troubleshooting:If slices are crumbly, the temperature is probably too cold; raise temperature ONE degree at a time
General Conditions/Settings:
~11 +/- millimeters/second
850 nanometers (thickness)
~6 degrees
See Louisa Howard about protocol for making glass knives