High Pressure Freezing and Immunoelectron Microscopy

Friday 09/12/2014

Looking for improved cellular preservation of ultrastructure for morphological studies and better antigen retention for immunolabeling work?   Look no further as the Electron Microscopy Resource offers the equipment needed to accomplish this. 

The Leica EM PACT2 is a high pressure freezing machine that is designed to rapidly freeze samples to liquid nitrogen temperature and simultaneously exposing them to high pressures (2100 bar).  It freezes samples up to 200um into the specimen without significant ice crystal damage.  Following high pressure freezing, the samples are put into a Leica EM AFS2 automatic freeze substitution apparatus.  Here the frozen sample is immersed into a fixative at low temperatures for 1-5 days replacing melting water molecules with fixative solution and fixing everything in place. Next, morphological studies or immunolabeling can be carried out after embedding in resin.

HIV capsid - Immunoelectron MicroscopyDr. Jaisri Lingappa uses these techniques in her research which is aimed at identifying cellular factors and viral-host interactions required for HIV capsid assembly.  The image is from Jaisri Lingappa’s laboratory from the following article:

Klein KC, Reed JC, Tanaka M, Nguyen VT, Giri S,Lingappa, JR 2011. HIV Gag-leucine zipper chimeras form ABCE1-containing intermediates and RNase-resistant immature capsids similar to those formed by wild-type HIV-1 Gag. Journal of Virology. 85(14):7419-35.