Protocol

Author

Lily Shen

Overview

A typical processing schedule for transmission electron microscopy (can also be used for light microscopy).

Units, terms, definitions

Transmission Electron Microscopy

Light Microscopy

DH2O – Distilled water

EtOH – Ethanol

Epon Araldite

Procedure

1. Dissect tissue immersed in a buffer* (the same buffer which you will use for fixation later). Best size of the sample is about 3mm x 3mm.
2. Primary fixation in 2.5% glutaraldehyde + 4% formaldehyde in 0.1M PBS for 2hrs.
3. Wash out Fixative in buffer (e.g. 0.1M PBS)* 3 x 10min changes
4. Post Fix in 1% OsO₄(made up in washing buffer or DH₂O) for 1 hr.
5. Wash out Fixative in DH₂O 3 x 10 mins changes.
6. Dehydration Series: 50% – 70% (sample can be left overnight at this stage) – 80% – 90% – 95% EtOH x 10mins each; 100% EtOH 10mins x 3 changes. 1 : 1 100% EtOH : Acetone for 10mins; 100% Acetone for 10mins.
7. Infiltration in
   • 2 : 1 100% Acetone : Epon Araldite for 30 mins
   • 1 : 1 100% Acetone : Epon Araldite for 30 mins
   • 1 : 2 100% Acetone : Epon Araldite for 30 mins
   • Pure Epon Araldite 3 x 2 hr changes (sample can be left overnight at any of these stages)
8. Embed in fresh resin** and cure overnight at 60^oC.

*NOTE: Choice of buffer e.g. PBS (Phosphate Buffered Saline) can be varied according to user. **NOTE: Choice of resin is also variable according to material intended for embedding and the desired end result.

Notes and trouble shooting tips

This protocol is only suitable for light microscopy and transmission electron microscopy, NOT microscope work involving immunolabeling.

Related protocols: Preparation of Material for TEM Examination

Health, safety & hazardous waste disposal considerations

Glutaraldehyde Formaldehyde