This page is under construction (09/12/2022).
Based on a protocol from "Experimental Neuroanatomy" by J. P. Bolam (1992) Oxford University Press.
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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 (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
- In 150 ml dH2O, add
- 0.55 g NaH2PO4•H2O (or 0.478 g NaH2PO4)
- 1.03 g Na2HPO4•7H2O (or 0.545 g Na2HPO4)
- Adjust pH to 7.4.
- Add dH2O to 160 ml
- Add 120 ml ethylene glycol
- Add 120 ml glycerol
- Mix well. Store this solution at 4°C
Tissue Storage Procedure
- 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.
- Place the vials at 4°C until the tissue equilibrates and stays in anti-freeze. (usually takes overnight)
- 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
- transfer the tissue into 0.1M phosphate buffer and wash 10 min x 3
- you may want to dissect out regions of interest (and embed into 9% agarose if thickness is 100 um or less) at this point
- transfer the tissue into 0.1M sodium cacodylate buffer and wash 10 min x 3
- proceed with heavy metal staining
Freezer Inventory