Microwave-assisted Chemical Fixation of Acute Hippocampal Slices

0.  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.

• You must be trained to break open glass ampules.
• 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)
  • Sabatini DD, Bensch K, Barrnett RJ (1963) Cytochemistry and electron microscopy - The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J Cell Biol 17:19-58. (PDF)
    

1.  Reagents and supplies:

• For fixative solution: 
  • A 500-ml graduated cylinder (This cylinder should be dedicated for use with aldehydes.)
    
  • Pasteur pipette and bulb
  • 1-ml micropipet and tips
    
  • Erlenmeyer flasks, beakers, stir bars
    

  • 5-ml cryo-tubes (e.g., ThermoScientific 5000-0050) labeled with date and contents (ask Masa for the labels)
    

  • purified water (e.g., Ricca Chemical 915025 or Fisher 9150-25)
    

  • sodium cacodylate trihydrate (solid; Ladd Research 20305)

  • glutaraldehyde (70% aqueous solution in 10-ml ampules stored at 4°C; Ladd Research 20108)

  • formaldehyde (20% aqueous solution in 10-ml ampules stored at 4°C; Ladd Research 20300)

  • calcium chloride dihydrate (CaCl₂·2H₂O)

    • Make a 0.4 M stock solution by adding 1.176 g into 20 ml of purified water. This can be stored in a glass vial at 4°C.
      

  • magnesium sulfate heptahydrate (MgSO₄·7H₂O)

    • Make a 0.8 M stock solution by adding 3.944 g into 20 ml of purified water. This can be stored in a glass vial at 4°C.
      
  • pH meter (with calibration standards)
    
  • 1N NaOH (aq) for adjusting pH
    
  • 1N HCl (aq) for adjusting pH
    

• For microwave-assisted fixation:

  • 1.5 tube per slice of 6% glutaraldehyde/2% formaldehyde (stored at -20°C in the freezer in NHB 3.360E)

  • tri-pour beaker

  • 6-well plate or 35 mm plastic Petri dish (12-well plate can be used for mouse slices)
    

  • 4 rings (used to make nets for interface chamber)

  • microwave oven with flatbed (no turn table)

    • Exhaust must be properly vented → see below for how to set it up.
      

    • We currently use a consumer model with 1500 W output, but also used one with 700 W output previously (Jensen & Harris, 1989)

  • an array of neon bulbs (e.g., EMS 97036-01)
    

2. Setting Up a Microwave Oven

1. Place the microwave oven as close to your electrophysiology rig as practical.
2. Properly vent the exhaust by either connecting vent of your microwave oven to an exhaust vent duct in your room or placing it in a fume hood.
3. Locate the hot spots:
   1. Run microwave empty for ~30 seconds to warm up the magnetron.
   2. Place a neon bulb array in microwave and run it for 30 sec.
      
   3. Observe which of the bulbs are lighting up, indicating hot spots. Repeat to cover the entire floor of the microwave chamber. Mark the cold spot.
4. If there is not a cold spot, or if it is too small to cover your dish or well plate, add a water load (water in 500-ml bottles; see image below) to absorb reflected irradiation. Check for hot spots again.
   • 
5. Place a dish or well plate filled with water wormed to 31°C in a cold spot. Run the microwave for 20 sec. Check temperature of the water to make sure it is below 50°C.

3.  Fixative Solution

1. Remove 70% glutaraldehyde from refrigerator (and leave at RT) 3 days prior to fixative preparation.
2. In an Erlenmeyer flask, dissolve sodium cacodylate, calcium chloride, and magnesium sulfate into purified water. See the table below for the amounts.
3. Adjust pH to 7.4 with 1N NaOH or 1N HCl.
4. Move the solution to a fume hood before adding the aldehydes. Keep stirring the solution.
5. Use a Pasteur pipet (or a narrow disposable pipet) to dispense 70% glutaraldehyde. Rinse the ampules with small amounts of the solution to remove as much glutaraldehyde as possible.
6. Add formaldehyde to the flask.
7. Once everything is completely dissolved, use a graduated cylinder to bring to the final volume to 350 ml with purified water.
8. Dispense the fixative into 5-ml cryo tubes (labeled with date and contents; you need about 70 tubes for the recipe below), and store at -20°C.

4.  Microwave-assisted chemical fixation

1. Approximately 15-20 minutes before the end of your experiment, begin preparing for fixation.

2. Take 1.5 tube per slice of the fixative solution out of the freezer in NHB 3.360E.

3. Place tubes in a tri-pour beaker, place beaker in sink, and allow warm water to run over the tubes to thaw the fixative (should be warmed to 31°C).

4. Grab a 6-well plate on the shelf above the vibratome and 4 glass rings (ring only, no net) from the net-making supply bucket. Label the wells for the slices to be fixed.
   

5. Place each of the 4 rings in a separate well of the 6-well plate.

6. 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 microwave to 20 seconds.

   1. Time should be adjusted depending on the method of subsequent tissue processing.

7. Once fixative is warmed, add fixative with a transfer pipette to each of the 4 wells (one 5-ml tube per well).

8. After last data point is collected and the recording ends, remove the camera/caps, withdraw all of the electrodes out of the chambers and fully retract manipulators.

9. Bring 6-well plate with fixative over to the rig and starting with the first chamber, take off the cover, pick up net by edge using ephys tweezers, and then with help of fix tweezers (marked with red tape), flip the net over onto ring in 6 well plate so that slice is completely submerged, but not touching the bottom of the well.

10. Repeat step 9 with remaining 3 chambers. Note to be very CAREFUL of recording electrodes.

11. Place 6-well plate with all 4 slices in microwave, close door and press start.

12. After microwave is done, take 6-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.

    1. The fixed tissue should be vibrasliced on the following day.
       
13. Place empty tubes and any other waste in solid waste bag in fume hood in NHB 3.360E or 3.360H.

5.  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.

• Sharps: Pasteur pipettes must be discarded into a sharps container labeled "Pb-aldehydes"