Safety precautions
This procedure involves the use of hazardous chemicals (including embryotoxins). Review MSDS for information on exposure limit, health risks, first aid, handling, etc.
Perform this procedure only in the designated area.
Wear appropriate Personal Protective Equipment for this procedure:
Lab coat
Nitrile gloves (double-layer; regularly check for holes)
Eye goggles
Mask (optional)
Plastic apron (optional)
Shoulder-length gloves (optional)
Place a piece of absorbent sheet on the work surface before starting the procedure.
Have all waste containers ready (see Clean-up).
- Read the following papers:
- Feinberg MD, Szumowski KM, Harris KM (2001) Microwave fixation of rat hippocampal slices. In: RT Giberson, RS DeMaree Jr. (eds.) Microwave Techniques and Protocols. Humana Press: Totowa, New Jersey. pp. 75-88. (PDF)
- Jensen FE, Harris KM (1989) Preservation of neuronal ultrastructure in hippocampal slices using rapid microwave-enhanced fixation. J. Neurosci. Methods. 29:217-230. (PDF)
- Karnovsky MJ (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137A-138A. (PDF)
Reagents and supplies:
For fixative solution:
15-ml centrifuge tubes
purified water
sodium cacodylate (Ladd Research)
glutaraldehyde
paraformaldehyde
calcium chloride (CaCl2)
magnesium sulfate (MgSO4)
For microwave-assisted fixation:
2 tubes of 6% glutaraldehyde/2.5% paraformaldehyde (stored in the freezer in NHB 3.360E)
tri-pour beaker
12-well plate
4 rings (used to make nets for interface chamber)
microwave oven (exhaust must be properly vented)
an array of neon bulbs
Microwave-assisted chemical fixation
Approximately 15-20 minutes before the end of your experiment, begin prepping for fixation.
Take 2 tubes of 6% glutaraldehyde/2.5% paraformaldehyde out of the freezer in the processing room.
Place tubes in a tri-pour beaker, place beaker in sink, and allow warm water to run over the tubes to thaw the fixative.
Grab a 12-well plate on the shelf above the vibrotome and 4 rings (ring only, no net) from the net-making bucket.
Place each of the 4 rings in a separate well of the 12-well plate.
Place neon bulb array in microwave, and run microwave for ~ 30 seconds to warm up the magnetron. Make sure none of the bulbs light up (which indicates a hot spot). Take array out of microwave and leave door ajar. Set timer to 20 seconds.
Once fixative is thawed, add ~ 2 ml of fixative with a transfer pipette to each of the 4 wells (basically one tube’s worth).
After last data point is collected and the Synchrobrain program ends, push the cameras out of the way, withdraw all of the stimulating and recording electrodes out of the chambers and fully retract manipulators.
Bring 12-well plate with fixative over to rig and starting with the first chamber, take off the cover, pick up net by edge using tweezers (marked with red tape), flip net over onto ring in 12 well plate so that slice is completely submerged, but not touching the bottom of the well.
Repeat step 9 with remaining 3 chambers. Note to be very CAREFUL of recording electrodes.
Place 12-well plate with all 4 slices in microwave, close door and press start.
After microwave is done, take 12-well plate out and add remaining tube of fixative to all 4 wells. Place lid back on plate and leave in the fume hood overnight.
- Place empty tubes and any other waste in solid waste bag on top of microwave.
Clean-up
Waste containers:
Hazardous Liquid Waste: Pour all waste into the proper waste collection bottles available in the fume hood in NHB 3.360E.
Aldehyde-Cacodylate (fixative solution, cacodylate buffer)
Hazardous Solid Waste: Place all contaminated solid waste (e.g., gloves, tubes, processing dishes, etc.) into hazardous waste bags in the fume hood. All tubes must be uncapped.