Use of antibodies from the same host species in double labeling immunofluorescence on trypanosome cytoskeleton

Several molecular tools are currently available to study trypanosomes however, there are some limitations regarding commercial monoclonal and polyclonal antibodies assortment. Here, we demonstrate a double labeling immunofluorescence to study Trypanosoma brucei cytoskeletal proteins employing same host species primary and secondary antibodies with different IgG subclasses. Immunofluorescence was performed incubating first with mouse polyclonal antibody (IgG) produced in our laboratory and with the secondary mouse IgG antibody conjugated to Alexa 594. Thereafter, a blocking step with rabbit anti-mouse IgG antibody was required before mouse monoclonal antibody (IgG1 or IgG2) was added followed by the addition of the specific secondary IgG antibody conjugated to Alexa 488. Thus, this method can be adapted to other eukaryotes when the source of primary antibodies is limited.