Biology Reference
In-Depth Information
and embedded at low temperatures to minimize extraction and
better preserve the native structure of the samples, including
immunogenic characteristics for labeling (
see
Fig.
3b
). Specific
materials needed for freeze substitution are:
(a) Fixative: Paraformaldehyde 4 % in PBS (protect from
light).
(b) Leica EM CPC cryofixation system.
(c) Leica EM AFS2 freeze substitution and low temperature
embedding system.
(d) Embedding medium: Lowicryl HM20 embedding kit
(Agar Scientific cat#R1034).
(e) En bloc staining and dehydration agent: 0.5 % Uranyl
acetate in dried methanol (protect from light and store
at −20 °C).
The general procedure is similar to that of Epon embed-
ding, with the appropriate reactive changes to carry out a
milder chemical fixation and dehydration. After chemical fixa-
tion, samples are treated with a cryo-protectant like glycerol to
prevent the formation of ice crystals in the samples during
freezing. Cell pellets are flash-frozen in the Leica EM CPC by
plunging in liquid ethane. Then, the samples are transferred to
the freeze-substitution unit (Leica AFS) for low temperature
dehydration, embedding, and polymerization with UV light.
The protocol is summarized in Table
1
, and the details of AFS
programming are given in Table
2
.
Table 1
Summary of freeze-substitution protocol
Step
Reagent
Time
Temperature
Fixation
Paraformaldehyde 4 %
in PBS
10 min
Room temperature
Cryo- protection
Glycerol 15 % in PBS
15 min
4 °C
Glycerol 30 % in PBS
15 min
4 °C
Cryo-fixation by plunging
Staining
Uranyl acetate 0.5 %
in methanol
Change twice daily during 3 days
−90 °C
Wash
Methanol 100 %
1 h, repeat three times
−40 °C
Embedding
Methanol: Resin (3:1)
1 h
−40 °C
Methanol: Resin (1:1)
1 h
Methanol: Resin (1:3)
1 h
Resin 100 %
15 min
Resin 100 %
Overnight
Resin 100 %
4 h
Resin 100 %
48 h
