ELECTRONMICROSCOPY

E.M. PROCESSING SCHEDULE - EPOXY RESIN

1. Fix tissue in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer at 4^oC, for a minimum of 4 hours. Tissue should be cut into approx. 1mm cubes for fixing. This may be done in a drop of fix on a sheet of dental wax, using a razor blade. Place in glass processing vials and close with plastic caps. (Tissue may be stored at this stage.)

2. Wash in 0.1M buffer - 1 hour x 2. (Or overnight at 4^oC.)

3. Post fix in osmium tetroxide in 0.2M buffer - 1 hour. (Mix equal quantities of 2% aqueous OsO4 and 0.4M buffer and use immediately.)

4. Rinse in 0.2M buffer - 5 mins. x 2.

5. Dehydrate in 70% ethanol - 20 mins. x 2. (May be stored overnight at this stage if absolutely necessary.)

6. Dehydrate in 90% ethanol - 10 mins. x 2.

7. Dehydrate in 100% ethanol - 20 mins. x 2.

8. Propylene oxide (1.2 epoxy propane) - 10 mins. x 2.

9. Propylene oxide/epoxy resin mixture (50/50) - 1 hour.

10. Epoxy resin - overnight - with caps removed from vials. (Allows any remaining propylene oxide to evaporate.)

11. Embed in labelled capsules with freshly prepared resin.

12. Polymerise at 60^oC - 48 hours.

NOTES

• All steps must be performed in a fume cupboard and gloves should be worn throughout.

• Osmium tetroxide, propylene oxide and propylene oxide/resin waste should be collected in bottles for safe disposal.

• Steps 2 to 11 - processing vials should be on a rotating mixer.

• Times at steps 10 and 11 need to be prolonged for tough specimens such as skin, tendon etc.. 2 hours at 10 and several more hours at 11.

• For very urgent specimens processing times may be reduced as long as the blocks of tissue are very small. Polymerisation can be achieved in 1 hour at 100^oC using gelatine capsules (the polythene ones melt at this temperature). Blocks must be cooled in water and are ready to cut in 15 mins..

E.M. PROCESSING SCHEDULE - ACRYLIC RESIN

1. Fix tissue in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer at 4^oC, for a minimum of 4 hours. Tissue should be cut into approx. 1mm cubes for fixing. This may be done in a drop of fix on a sheet of dental wax, using a razor blade. Place in glass processing vials and close with plastic caps. (Tissue may be stored at this stage.)

2. Wash in 0.1M buffer - 1 hour x 2. (Or overnight at 4^oC.)

3. Post fix in osmium tetroxide in 0.2M buffer - 1 hour. (Mix equal quantities of 2% aqueous OsO4 and 0.4M buffer and use immediately.)

4. Rinse in 0.2M buffer - 5 mins. x 2.

5. Dehydrate in 70% ethanol - 20 mins. x 2. (May be stored overnight at this stage if absolutely necessary.)

6. Dehydrate in 90% ethanol - 10 mins. x 2.

7. Dehydrate in 100% ethanol - 20 mins. x 2.

8. LR White resin - overnight.

9. LR White resin - 1 hour.

10. LR White resin - 1 hour.

11. Embed in closed, labelled, gelatine capsules with fresh resin.

12. Polymerise at 60^oC - 24 hours. (Polymerise at 50^oC for immunocytochemistry.)

NOTES

• All steps must be performed in a fume cupboard and gloves should be worn throughout.

• Osmium tetroxide/buffer waste should be collected in bottles for safe disposal. Resin waste may be polymerised.

• Steps 2 to 10 - processing vials should be on a rotating mixer.

• If results are ugently required steps 8-10 may be shortened by performing 4-6 changes of resin at 60^oC over 3 hours.

• If polymerisation using the accelerator is necessary step 3, osmium tetroxide, must be omitted or artefact will occur due to overheating. Step 3 should also be omitted if immunocytochemistry is to follow. Resin which is nearing its “use by” date should not be used with OsO4 as some polymerisation may occur during step 8.

PRIMARY FIXATIVE

2.5% glutaraldehyde in 0.1M sodium cacodylate buffer.

Add 1ml of 25% glutaraldehyde stock to 9mls of buffer.

Best prepared and used fresh.

BUFFERS

The pH should be within the range 7.2 - 7.4.(Corrected with 0.1M HCl.)

0.1M sodium cacodylate - 10.7g in 500mls of distilled water.

0.2M sodium cacodylate - 21.4g in 500mls of distilled water.

0.4M sodium cacodylate - 42.8g in 500mls of distilled water.

EPOXY RESIN (AGAR 100^®)

(Supplier: Agar Scientific, 66A Cambridge Road, Stansted Essex, CM24 8DA, U.K.)

Agar 100 resin - 24g.

DDSA (E.M. grade) - 13g.

MNA (E.M. grade) - 13g.

BDMA - 1ml.

Mix thoroughly in a disposable beaker using a wooden spatula. (May be stored in the freezer compartment of a refrigerator for short periods if tightly sealed.)

ACRYLIC RESIN (LR WHITE^®)

(Supplier: London Resin Company, P.O. Box 2139, Reading, Berkshire, RG7 4YG, U.K.)

Use straight from the bottle unless the results are needed urgently in which case the accelerator may be used. The resin will polymerise in approximately 10 minutes using a mixture of 1 drop of accelerator to 10ml of resin. The capsules should be stood on ice, or put in the ice box of a refrigerator, during this time as excessive heat is produced by the reaction.

Osmium tetroxide should not be used in conjunction with the accelerator or if immunocytochemistry is to follow.

STAINS (Impregnation with heavy metals)

Uranyl acetate:

Methanolic - saturated uranyl acetate in 50% methanol.

Aqueous - saturated uranyl acetate in distilled water.

(Keeps for approx. 3 months - store in a brown glass bottle.)

Reynold's lead citrate:

1.33g lead nitrate.

1.76g sodium citrate.

30mls distilled water.

• Shake for 1 minute.

• Allow to stand for 30 mins. shaking the solution occasionally.

• Add 8mls 1M NaOH (Analar) and mix.

• Dilute to 50mls with distilled water.

• Final pH should be pH12. (Keeps approx. 6 months)

TIMING OF STAINS FOR EPOXY RESIN

Uranyl acetate:

• 10 mins. for methanolic.

• 20 mins. for aqueous.

Lead citrate:

• 5 mins.

TIMING OF STAINS FOR ACRYLIC RESIN

Uranyl acetate:

• 5 mins. Use aqueous ONLY (alcohol softens the resin).

Lead citrate:

• 5 mins.

NOTES:

• All staining solutions should either be filtered through Millipore filters or centrifuged before use.

• Use filtered distilled w