Biology Reference
In-Depth Information
4. EM-grade 25 % glutaraldehyde. After opening the vial, store at
4 °C. Glutaraldehyde can irritate the skin and mucous mem-
brane by direct contact or vapor inhalation. It must always be
handled in a fume hood.
5. Tannic acid.
6.
En bloc
fixative/staining agent: 1 % osmium tetroxide (OsO
4
),
0.8 % potassium ferricyanide (K
3
[Fe(CN)
6
]) in water. Prepare
the mixture on the same day and protect from light. Caution:
OsO
4
must always be handled in a fume hood and skin contact
must be avoided at all times.
7.
En bloc
staining agent: uranyl acetate 2 % w/v (protect from
light). Follow safety measures as described in previous
sections.
8. Dehydration agent: dried acetone.
9. Embedding medium: 812 Epon Embedding Kit (Electron
Microscopy Sciences). Caution: some of the kit components
are irritating to the eyes and respiratory tract; others are cor-
rosive and flammable. Do not get into eyes, on skin, or cloth-
ing. Use only with adequate ventilation. Do not breathe vapor.
Follow the MSDS instructions for each kit component.
For 10 mL of Epon, weigh:
(a) 812 resin: 4.8 g.
(b) DDSA (Dodecenyl Succinic Anhydride): 1.9 g.
(c) MNA (Methyl Nadic Anhydride): 3.3 g.
(d) Accelerator BDMA (Benzyl Dimethylamide): 5 drops
(from a 3 mL plastic Pasteur pipette).
All these substances are dense liquids. To weigh them, use
a 3 mL plastic Pasteur pipette to add gradually the resin to a
Falcon tube placed on the scale. Mix well. Centrifuge the mix-
ture before using it to remove air bubbles (15,900 ×
g
for
10 min).
10. Wooden toothpicks.
11. Encapsulation: Beem capsules size 3 (Electron Microscopy
Sciences).
1. Diamond knife (Diatome 45°).
2. Ultramicrotome (Leica EM UC7 or similar).
3. Single edge blades (TAAB cat#B056 or equivalent).
4. Wire loop (Diatome Perfect Loop, Electron Microscopy
Sciences).
5. Glass knife maker (e.g., RMC Products GKM-2 or similar).
6. Glass knife strips (Electron Microscopy Sciences).
7. Needles (1.2 × 40 mm 18G).
2.6.2
Sectioning
