Chapter 5 - Special Methods

Summary

INTRODUCTION

Because the following methods are used frequently in an EM laboratory (especially a clinical laboratory), it was decided to include them in a separate chapter. The list of chemicals and equipment at the beginning of each technique includes only those unique to each method since general supplies are covered in previous chapters, as are methods for preparing solutions.

CULTURED CELLS

Chemicals and Equipment

• Thermanox coverslips.

• Polypropylene medicine cups – 30 mL.

Method

1. Fix coverslips containing cell monolayer in 2.5% glutaraldehyde in O.lmol/L cacodylate bufffer for 30 minutes.

2. Transfer each coverslip to a separate polypropylene medicine cup.

3. Rinse with several changes of buffer.

4. Postfix, dehydrate, and infiltrate with resin in the usual manner (see Chapter 2).

5. Place each coverslip into an embedding mold so that the monolayer is perpendicular to the plane of sectioning (for cross sections). If en face sections are required place the coverslip cell-side down on the surface of a blank, polymerized block of resin to which a small drop of fluid resin has been applied.

6. Cure in a 70°C oven for 6–8 hours or overnight.

7. Remove from the oven and allow to cool 20 minutes.

8. Strip off the coverslip (if embedded en face) with a pair of forceps and trim and cut the block.

Notes on Method

• If cells have been grown on glass coverslips or in a polystyrene culture flask, the monolayer must be separated by enzymatic digestion or by scraping with a “rubber policeman,” centrifuged, and encapsulated (see Fine Needle Aspiration Biopsies). […]