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Electron Microscopy Tissue Preparation

Solid Tissue Surgical Specimens

1. As soon as possible after the tissue has been removed from the blood supply, and preferably in the operating suite, place the tissue on an index card or piece of dental wax.

2. Cover the tissue with a couple of drops of cold (4º C) 4% buffered glutaraldehyde fixative, and cut into blocks no larger than 1 mm3 with a sharp, clean razor blade.

3. Place the tissue blocks in about 5 cc cold fixative, and put in a refrigerator for a minimum of 2 hours, or until the next morning. The tissue may also be sent directly to the EM lab for storage and processing.

4. When handling tissue for electron microscopy, caution must be exercised not to squeeze it. Manipulate the tissue using the fixative as a suspension medium and maintain a 1.5 mm distance between the tips of forceps used. Alternatively, a pipette may be used to transfer the blocks, if they are small enough to fit in the pipette.

Solid Tissue other

1. Fresh tissue received by the Pathology laboratory should be cut into mm3 blocks, then placed into a covered container with 4% buffered glutaraldehyde at 4º C for at least two hours.

2. After two or more hours place the tissue in 0.1M sodium cacodylate buffer at 4º C. for at least 15 min. up to a week.

The remainder of this procedure may be performed in a covered 5 ml. glass container in a refrigerator and/or on a rotating stand or in a RMC EMP 5160 Ultraprocessor Tissue Processor .

3
. Make three changes of 0.1M sodium cacodylate buffer for at least 15 min. each at 4º C.

4. Place in a covered container containing 2% osmium tetroxide in 0.1M sodium cacodylate buffer for one hour at 4º C.

The remainder of this procedure may be done in an uncovered container at room temperature.

5. Place in 0.1M sodium cacodylate buffer for 5 min..

6. Place in in 50% ethanol/distilled water for 5 min.

7. Place in in 70% ethanol/distilled water for 10 min.

8. Place in in 90% ethanol/ distilled water for 10 min.

9. Place in three changes of 100% reagent grade ethanol, 20 min. each.

10. Place in 50% L. R.White/100% reagent grade ethanol for 30 min.

11. Place in 75% L. R. White/100% reagent grade ethanol for 30 min.

12. Place in two changes in 100% L. R. White, 30 min. each.

13. Embed in L. R. White in covered gelatin capsules and allow to stand
overnight at room temperature in a fume hood to ensure complete infiltration.

14. Place in a 60º C oven for 22 to 26 hours.

 

reviewed 8-24-06 CPD

Last Updated: 10/2/08