Leica Microsystems EM AFS2 Application Note

Leica Microsystems
Brand
Leica Microsystems
Model
EM AFS2
Type
Application Note

Leica Microsystems EM AFS2

The Leica Microsystems EM AFS2 is a high-pressure freezer and freeze substitution system that allows for the preservation of biological samples for electron microscopy. The EM AFS2 uses a combination of high pressure and low temperature to rapidly freeze samples, which helps to prevent the formation of ice crystals that can damage the sample. The EM AFS2 also uses a freeze substitution process to replace the water in the sample with a solvent that is compatible with electron microscopy. This process helps to preserve the ultrastructure of the sample and makes it possible to obtain high-resolution images.

Leica Microsystems EM AFS2

The Leica Microsystems EM AFS2 is a high-pressure freezer and freeze substitution system that allows for the preservation of biological samples for electron microscopy. The EM AFS2 uses a combination of high pressure and low temperature to rapidly freeze samples, which helps to prevent the formation of ice crystals that can damage the sample. The EM AFS2 also uses a freeze substitution process to replace the water in the sample with a solvent that is compatible with electron microscopy. This process helps to preserve the ultrastructure of the sample and makes it possible to obtain high-resolution images.

Application Note
Sample Preparation of Mouse heart
related instrument Leica EM AFS2
Material
Research
Life Science
Research
Medical
Research
Industrial
Manufacturing
Natural
Resources
2
Immuno - Electron Microscopy of high
pressure frozen and freeze substituted
mouse heart
COURTESY
PROTOCOL HPF - AFS MOUSE HEART IEM:
Mouse heart tissue from wild-type (WT) and αT -catenin KO (KO) mice was excised, immersed in 20% (w/v) BSA
and frozen immediately in a high-pressure freezer (EM PACT; Leica Microsystems, Vienna,Austria). Freeze subs-
titution was carried out using a Leica EM AFS (Leica Microsystems) in dry acetone containing 2% ddH2O , and
0.1% glutaraldehyde over a 4-days period as follows:
-90°C for 24 hours, 2°C per hour increase for 15 hours, -60°C for 24 hours, 2°C per hour increase for 15 hours, and
-30°C for 24 hours. Samples were then washed 3 times in pure acetone between -30°C and 0°C and slowly war-
med up to 4°C, infiltrated stepwise over 3 days at 4°C in LR-White and embedded in capsules. The polymerization
was performed in Leica EM AFS using UV lamp over 6 days starting at 20°C and ending at 37°C.
Ultrathin sections were made using an ultramicrotome (Leica EM UC6) and post-stained in in a Leica EM AC20 for
40 min in uranyl acetate at 20 °C and for 10 min in lead citrate at 20 °C.
Grids were viewed with a JEM 1010 transmission electron microscope (JEOL, Tokyo, Japan) operating at 80 kV
using Image Plate Technology from Ditabis (Pforzheim, Germany).
Immunolabeling and label quantification were performed as described previously (Goossens et al., 2007).
The following primary antibodies were used for immuno-EM:
Cx43-polyclonal rabbit (1:50; Sigma) and Desmin polyclonal rabbit (1:50; AbCam).
In the article of Li et al. also other images are shown, processed (with EM Leica EM-Pact and Leica AFS) for spurr‘s
resin and HM20 (with HPM010), but that‘s no problem, the protocol we explain here is for the shown images.
For details see: Li et al., J Cell Sci 125, 1058-1067. , 2012 (http://jcs.biologists.org/content/125/4/1058.long)
Riet De Rycke
resp. DMBR-PSB Transmission Electron Microscopy - Core facility
VIB1 - Department for Molecular Biomedical Research
VIB2 - Plant Systems Biology
9000 Gent
LNT Application Note - Mouse heart 3
Single immunogold labeling of Cx43 with silver amplifi-
cation at representative gap junctions of the intercala-
ted disc (ICD) from αT-catenin KO mouse heart.
B
Single immunogold labeling of Cx43 with silver amplifi-
cation at representative gap junctions of the intercala-
ted disc (ICD) from WT mouse heart.
Single immunogold labeling of desmin in a desmosome
from αT-catenin KO mouse heart.
Single immunogold labeling of desmin in a desmosome
from αT-catenin KO mouse heart.
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Electron and Light Microscope. Excellent Sample Preparation is a
prerequisite for perfect microscopy. Your image starts here!
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Leica EM AFS2 Application Note Mouse Heart ∙ 07/2014 ∙ Copyright © by Leica Mikrosys-
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Leica Microsystems EM AFS2 Application Note

Leica Microsystems
Brand
Leica Microsystems
Model
EM AFS2
Type
Application Note

Leica Microsystems EM AFS2

The Leica Microsystems EM AFS2 is a high-pressure freezer and freeze substitution system that allows for the preservation of biological samples for electron microscopy. The EM AFS2 uses a combination of high pressure and low temperature to rapidly freeze samples, which helps to prevent the formation of ice crystals that can damage the sample. The EM AFS2 also uses a freeze substitution process to replace the water in the sample with a solvent that is compatible with electron microscopy. This process helps to preserve the ultrastructure of the sample and makes it possible to obtain high-resolution images.

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