Biology Reference
In-Depth Information
4. Appropriate buffer without dye traces (PBS, Tris-HCl,
HEPES, etc.).
5. Propidium iodide. To prepare a stock solution from the solid
form, dissolve PI (MW = 668.4) in deionized water or buffer at
1 mg/mL (1.5 mM) and store at 2-6 °C, protected from light.
Caution: PI is a potential mutagen and should be handled with
care. It must be disposed of safely and in accordance with
applicable local regulations.
6. Origin software for data analysis (
http://www.originlab.com
)
.
2.2 Negative
Staining Transmission
Electron Microscopy
1. Copper grids covered with carbon or carbon/collodion film
(for example GILDER GRIDS G400-C3, 400 lines/in. Square
Mesh Copper).
2. Glow discharge equipment (Emitech K100x or equivalent).
3. High precision grade tweezers (high precision grade Dumont
Tweezers type 4, stainless steel anti-mag or equivalent).
4. Whatman Grade Nº1 Filter paper, 90 mm diameter.
5. Purified adenovirus sample.
6. Staining agent: uranyl acetate. Usually prepared as a 1-2 % (w/v)
solution in double-distilled water without adjustment of the ini-
tial pH of approximate pH 4.3-4.5 (it precipitates above pH 5.0).
Uranyl acetate solutions must be protected from light and fil-
tered using 0.025 μm filters prior to use. Caution: uranyl acetate
has heavy metal solution waste category and requires following
local regulations for heavy metal handling, safety, and disposal. It
is toxic by ingestion and if inhaled as dust. Use of gloves and
safety goggles is required when preparing the diluted reagent.
7. Access to a 100/120 kV transmission electron microscope
(JEOL-JEM 1100 or similar).
2.3 Negative
Staining
Immunoelectron
Microscopy
of Purified Viruses
1.
Items 2
-
7
in Subheading
2.2
.
2. Carbon or collodion/carbon coated nickel EM grids (
see
Note 1
).
3. Tris-buffer saline (TBS: 10 mM TRIS-base, HCl up to pH 8.2,
150 mM NaCl).
4. Bovine serum albumin (BSA).
5. Cold water fish skin gelatin.
6. Blocking solutions: TBG: TBS + 0.1 % BSA + 1 % gelatin. TBS/
BSA: TBS + 1 % BSA.
7. Purified adenovirus sample.
8. Primary antibody of interest, as well as positive and negative control
antibodies. For example, an antibody against hexon can be used as
a positive control, and an irrelevant IgG as a negative control.
9. Secondary antibody conjugated to 10 nm colloidal gold par-
ticles (e.g., Goat Anti-mouse IgG + IgM EM-grade 10 nm cat#
25168 Electron Microscopy Sciences) (
see
Note 2
).
