Transmission Electron Microscopy – starting protocol for zebrafish

Almost all steps are done in small glass tubes (7ml) and on a slow rotating wheel (o/n steps can also be done in the cool room on a rotator).

Day1

1. anaesthetise fish and fix immediately in

            4% Glutaraldehyde

            1% PFA

            0.05M Cacodylate

            1mM MgSO4

            1% Sucrose

for about 1.5 h.

2. Depending on the part of the fish you want, try to cut it down after a initial fix of – say – 30min.

3. Wash 3x in 0.1 M Cacodylate buffer.

4. fix in 2% OsO4/0.1 M Cacodylate for 2 – 3h.

5. Wash in 0.1M Cacodylate buffer and keep at 4deg in the fridge o/n.

Day2

6. dehydrate with 50%, 70%, 80%, 90%, 96% and 100% EtOH over the course of one day. Change 100% EtOH again before keeping the specimen in the fridge o/n. (Can also stop at 90% and continue next day)

Day3

7. wash 2x with PPO

8. incubate in 1:1 Epon / PPO o/n.

Day4

9. change to 100% EPON

10 change EPON for another o/n incubation

Day5

11. embed and harden in 60degrees incubator (1-2 days)

Cut ‘semi-thin’ sections (1-5um) with glass knives on an ultramicrotome

Cut thin sections with a diamond knife

Stain sections using ‘standard EM stains’

 

 

TIMING OF STAINS FOR EPOXY RESIN

Uranyl acetate:

• 10 mins. for methanolic.

Lead citrate:

• 5 mins.
  
  

Epon resin

 

 

EPON RESIN

(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.)

 

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.

 

 

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)