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

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

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

see 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