Immune electron microscopy

Our antibodies (primarily J2) have been successfully used by multiple groups for the ultrastuctural localisation of dsRNA made by viral replication complexes in virus infected cells.

The group of Professor Snijder has shown how the Severe Acute Respiatory Virus (SARS)1 as well as the Equine Arteritis Virus (EAV)2  induce the formation of double membrane vesicles (DMVs) connected to the endoplasmatic reticulum, and has studied how these accumulate dsRNA as replication intermediates. Similar observations have been made by the Bartenschlager group for Dengue Virus3. Further references using our antibodies in immuno EM can be found in our publications database.

 

Immune electron microscopy image, where J2 antibody labeling shows abundance of dsRNA (black dots) in double-membrane vesicles (DMVs) of SARS-CoV infected Vero E6 cells, fixed at 7 hours post-infection. J2 was detected using immunogold conjugated to protein A. G: Golgi complex, M: mitochondria, N: nucleus. Figure taken from Knoops et al. (2008) PLoS Biol 6:e226.

 

 

 

The protocol used by the Leiden group in their latest publication on EAV2 for IEM visualisation of dsRNA in EAV-infected Vero E6 cells is given as an example of how our antibody can be used for this application:

The cells used in these studies were pre-fixed in with 3% paraformaldehyde in 0.1 M PHEM buffer [60 mM piperazide-1,4-bis (2-ethanesulfonic acid), 25 mM HEPES, 2 mM MgCl2, 10 mM EGTA] then cryofixed by High Pressure Freezing (HPF), followed by Freeze substitution (FS) in anhydrous acetone containing 0.2% uranyl acetate and 1% methanol at −90°C for 72 h. After this step the temperature was raised by 23.33°C/h to −20°C. The samples were kept for 1 h at this temperature, after which the temperature was lowered by 20°C/h to −50°C. After washing with ethanol, samples were infiltrated with Lowicryl HM20 (Electron Microscopy Sciences) and polymerized under UV light at −50°C. Thin sections were labeled with the anti-dsRNA antibody J2, which was detected using a bridging rabbit anti-mouse IgG antibody (DakoCytomation) and 10-nm protein A-gold particles. Grids, contrasted with uranyl acetate and lead citrate, were viewed in a Tecnai 12 BioTwin transmission electron microscope at 80 kV. 

1Knoops K, Kikkert M, Worm SH, Zevenhoven-Dobbe JC, van der Meer Y, Koster AJ, Mommaas AM, Snijder EJ. (2008) SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol 6:e226

2Knoops K, Barcena M, Limpens RW, Koster AJ, Mommaas AM, Snijder EJ. (2012) Ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral RNA synthesis. J Virol 86: 2474-2487

3Welsch S, Miller S, Romero-Brey I, Merz A, Bleck CK, Walther P, Fuller SD, Antony C, Krijnse-Locker J, Bartenschlager R. (2009) Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe 5:365-375

Additional information about our antibodies and their application can be found on our Frequently Asked Questions pages.