Biology Reference
In-Depth Information
11.
Fine-tipped tweezers with clamping ring to plunge samples in cryogen (Dumont
tweezers, Biology clamp)
12.
Rounded tweezers with insulator cover
13.
Primary antibody at saturating concentration in PBS containing 0.1% BSA
14.
Colloidal gold-coupled protein A (CMC Utrecht) or gold-coupled secondary
antibodies (Biocell or Aurion), gold diameter of 5 or 10 nm diluted in PBS
containing 0.1% BSA
15.
Ethane for fast freezing of the sample
16.
Liquid nitrogen for sample preparation and storage
17.
Cryovials with cap (2 ml volume)
18.
Thin metal rod and a spirit burner to prepare cryovials for frozen mice storage
19.
Domestic bleach (sodium hypochlorite) for digestion of organic material
20.
Triple-distilled water
21.
Pasteur pipettes with bent, melted tip to transfer replicas during cleaning/
washing
22.
Copper EM bare grids (400 mesh hexagonal, Gilder G400HEX-C3)
23.
Acetone to clean copper EM bare grids
24.
100% ethanol
25.
Filter paper
21.2
EQUIPMENT
1.
Microcentrifuge
2.
Spirit lamp
3.
Dissecting seeker
4.
Plunge-freezing unit (
Fig. 21.1
) (KF-80, former Reichert-Jung)
5.
Liquid nitrogen bench Dewar
6.
Liquid nitrogen storage tank (optional)
7.
Twin cold light source
8.
Freeze-etching unit (
Fig. 21.2
) (BAF 060; Bal-Tec)
9.
Specimen tables for freeze fracturing, modified to accommodate mica
rectangles (
Fig. 21.3
) (Bal-Tec, BU 02 098-T)
10.
Bal-Tec specimen table loading device to insert specimen tables and split mica
sandwiches under liquid nitrogen (
Fig. 21.4
)
11.
Porcelain spotting dishes (
Fig. 21.5
)
12.
TEM, preferentially with camera with tiling option (e.g., TemCam-F416,
TVIPS).
21.3
METHODS
In
Table 21.1
, the protocol is summarized and time estimates and points of possible
interruption are given.
