Long-term storage of chemically fixed vibratome-sectioned brain tissue

This page is under construction (09/12/2022).

Based on a protocol from "Experimental Neuroanatomy" by J. P. Bolam (1992) Oxford University Press.

Materials

• specimen container:

  • Larger brain: Fisher 03-337-7B

  • Smaller brain: Fisher 13-709-140; Fisher 03-388A

• purified water

• NaH₂PO₄•H₂O

• Na₂HPO₄•7H₂O

• pH meter, HCl, NaOH

• ethylene glycol

• glycerol

Anti-freeze Solution

for storing fixed tissue sections at -20°C (Reference: J.P. Bolam (1992) "Experimental Neuroanatomy"  Page 9.)

160 ml of 0.05M PB, pH 7.4 + 120 ml ethylene glycol + 120 ml glycerol

1. In 150 ml dH2O, add

   1. 0.55 g NaH₂PO₄•H₂O (or 0.478 g NaH₂PO₄)

   2. 1.03 g Na₂HPO₄•7H₂O (or 0.545 g Na₂HPO₄)

2. Adjust pH to 7.4.

3. Add dH₂O to 160 ml

4. Add 120 ml ethylene glycol

5. Add 120 ml glycerol

6. Mix well. Store this solution at 4°C

Tissue Storage Procedure

1. After vibratome-sectioning (using 0.1M phosphate buffer), transfer the sections into anti-freeze solution in vials. Tissue section may float on top → try to soak them in anti-Freeze so as not to get them dried out.

2. Place the vials at 4°C until the tissue equilibrates and stays in anti-freeze. (usually takes overnight)

3. Place the vials into a freezer at -20°C for long-term storage. (a freezer with manual defrost is preferred.)

Using Stored Tissue for EM Processing

1. transfer the tissue into 0.1M phosphate buffer and wash 10 min x 3

2. you may want to dissect out regions of interest (and embed into 9% agarose if thickness is 100 um or less) at this point

3. transfer the tissue into 0.1M sodium cacodylate buffer and wash 10 min x 3

4. proceed with heavy metal staining

Freezer Inventory