Immunogold Staining of M. tuberculosis

Immunogold staining was performed as described earlier (Sun et al., 2013 (link)) at the EM Facility of the James Hogg Research Centre (Saint Paul Hospital, Vancouver, BC, Canada). Briefly, M. tuberculosis-infected macrophages were fixed with 4% paraformaldehyde, dehydrated in graded series of ethanol and water, and infiltrated with LR White resin. After polymerization at 50°C, 60 nm sections were cut with a Leica EM UC6 microtome (Leica Microsystems, Switzerland) and collected on nickel grids. The samples were then stained with anti-Cpn60.2 antibody followed by labeling with colloidal gold conjugated anti-rabbit IgG. Sections were then post-fixed in 2% glutaraldehyde and subjected to silver enhancement with Silver R-Gent SE-EM (Aurion, Wageningen, Netherlands). Samples were then washed with distilled water, stained in 2% uranyl acetate, washed again, air dried and examined with a Tecnai 12 electron microscope (FEI Company, Hillsboro, OR, USA).