Immuno - Electron Microscopy of High Pressure Frozen and Freeze Substituted Mouse Heart
Application Note for Leica EM AFS2 - Life Science Research
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 Leica EM PACT (Leica Microsystems, Vienna, Austria).
Freeze substitution 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 warmed 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 Leica EM PACT and Leica EM AFS) for spurr‘s resin and HM20 (with Leica 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 2012 125: 1058-1067; doi: 10.1242/jcs.098640
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