Agarose Embedding and Vibraslicing Fixed Brain Tissue

under construction (09/20/2022)

Reagents and Supplies

• fixed tissue
• fine knife
• 9% agarose (low-melting temperature); melted and kept at 60-65°C
• embedding slides: Glass microscope slide with spacers glued on top. Spaces are small pieces of glass microscope slides. You need glass scoring pen and super glue.
  
  • A: spacer = thickness of one glass microscope slide
  • B: spacer = thickness of two glass microscope slides
• coverslips
• vibratome
• scalpel with a straight blade

Acute hippocampal slice tissue fixed after recording on MED-64

Step 1: At the end of electrophysiology recording, take an image of the slice in the MED-64 chamber/probe. Electrode contacts are 150 µm apart from each other in this example. Fix the slice using this protocol.

Step 2: After postfixation, take another image of the slice. Compare this image with the one from Step 1 to determine where to cut to excise the region of interest (red lines in area CA1 in this example). Look for landmarks such as net and harp indentations, pyramidal cell layer, hippocampal fissure, etc. Red dots indicate approximate location of electrode contacts.

Step 3: Use fine knife to dissect out the ROI (cuts indicated by red arrows).

Step 4: Place the excised tissue on Embedding Slide A

Step 5: Add a drop of melted 9% agarose.

Step 6: Place a coverslip and wait until agarose solidifies.

Step 7: Remove the tissue in agarose, flip it over, and place it on Embedding Slide B.

Step 8: Add another drop of melted 9% agarose.

Step 9: Place a coverslip and wait until agarose solidifies.

Step 10: Remove the embedded tissue from Embedding Slide B and cut into a trapezoid block as shown in the diagram below. Note the orientation of the tissue.

Step 11: Glue the agarose block containing tissue to the vibratome stage with the subiculum side down. Collect sections at 150 µm thickness. Because section thickness is the same as electrode pitch, each section should correspond to one column of electrode contacts.

Step 12: Take images of each vibratome sections. Compare them with the MED-64 image (Step 1) to locate first section corresponding to the electrode contacts closest to CA3. Check all vibratome sections to make sure they correspond with electrode locations.

Perfusion-fixed brain