Biomedical Engineering Reference
In-Depth Information
1 × 2-mm slot grids, 1 × 2-mm slot grids covered with formvar
and carbon (Electron Microscopy Sciences, Hatfi eld, PA, or
Agar, UK).
5. Whatman number 1 filter paper (Fisher Scientific,
Pittsburgh, PA).
1. 150 mM NaCl.
2. 10× PBS.
3. Dulbecco's modifi ed Eagle's medium (DMEM) supplemented
with 10 % fetal calf serum (FCS) and 2 mM l-glutamine (Gibco
BRL, Life Technologies).
4. HEPES buffer (0.2 M). Dissolve 4.77 g HEPES in 100 ml
distilled water, and add of 1 N HCl droplet by droplet to pro-
vide a pH of about 7.2-7.3
5. PBS-BSA buffer: 20 mM phosphate, 150 mM NaCl, pH 7.4;
1 % bovine serum albumin (BSA), optional, may reduce back-
ground, 0.5 M NaCl, 0.05 % Tween 20.
6. Fixative 1. 0.05 % glutaraldehyde plus 4 % formaldehyde in
0.15 M HEPES (pH 7.2-7.3). Dissolve 8 g paraformaldehyde
powder in 50 ml 0.2 M HEPES buffer, stirring and heating the
solution to 60 °C. Add drops of 1 N NaOH to clarify the solu-
tion. Add 1.25 ml 8 % glutaraldehyde and 50 ml 0.2 M HEPES
buffer. Dilute twice before use.
7. Fixative 2. 4 % formaldehyde in 0.15 M HEPES (pH 7.2-7.3).
Dissolve 4 g paraformaldehyde powder in 100 ml HEPES buf-
fer, stirring and heating the solution to 60 °C. Add drops of
1 N NaOH to clarify the solution.
8. Blocking solution. Dissolve 0.50 g BSA, 0.10 g saponin, 0.27 g
NH4Cl in 100 ml of 0.2 M HEPES (PH 7.2-7.3).
9. Gold enhancement mixture. Using equal amounts of the four
components (Solutions A, B, C, and D from a gold enhance-
ment kit), prepare about 200
2.2 Solutions
l of reagent per Petri dish (a
convenient method is to use an equal number of drops from
each bottle). First, mix Solution A (enhancer; green cap) and
Solution B (activator; yellow cap). Wait for 5 min, and then
add Solution C (initiator; purple cap) and fi nally Solution D
(buffer; white cap). Mix well. Development starts with the
addition of Solution C (initiator), so apply to sample as soon as
possible after adding C and D to minimize auto-nucleation
background. The development is not highly light sensitive, so
it may be conducted under normal room lighting. Secondary
GEEM solution: for slower development, substitute the fol-
lowing buffer for Solution D (buffer), 0.05 M sodium phos-
phate, 0.1 M NaCl, and pH 6.1.
μ
