A single computed slice through a tomographic reconstruction of a virological synapse formed between naturally HTLV-1 infected CD4+ T cells and autologous uninfected CD4+ target cells. HTLV-1 particles in the synaptic cleft were identified by immunolabeling using anti-Gag p19 labeling and peroxidase-DAB detection. This image has been downsampled from the raw data image which can be accessed using the link provided to the Cell Centered Database. For more information see: Majorovits et al. (2008) Human T-Lymphotropic Virus-1 visualized at the virological synapse by electron tomography. PLoS ONE 3: e2251, doi:10.1371/journal.pone.0002251, PMID 18509526
CD4+ T cells were isolated and cultured overnight to allow spontaneous expression of HTLV-1 proteins, and subsequently prepared for EM. To detect HTLV-1 Gag matrix protein in infected cells the samples were labelled against HTLV-1 Gag p19 (Tanaka et al., 1986) using the DAB kit from Molecular Probes (Invitrogen Ltd, Paisley, UK ). After the PFA/glutaraldehyde fixation, the samples were incubated in PBS containing 1% BSA, 0.1% Triton X-100 and 2% H2O2, for 1 h at 37uC to block non-specific antibody binding and to quench the endogenous peroxidase activity of the cells. To detect the HTLV-1 complexes, the cells were incubated with 5 mg/ml of anti-Gag p19 (GIN7 mAb)  diluted in PBS 1% BSA, for 1 h at 37uC. Then the second antibody, anti-Mouse mAb conjugated to HRP (Invitrogen Ltd, Paisley, UK ), was added at a final dilution of 1 mg/ml, and incubated for 30 minutes at 37uC. The cells were washed three times with PBS containing 1% BSA between each step. For detailed methods of the preparation, refer to the CCDB link provided.
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