Pleiotropic Effect of Hormone Insulin-Like Growth Factor-I in Immune Response and Pathogenesis in Leishmaniases

Figure 4

Detection of IGF-I within RAW 264.7 macrophages following infection with Leishmania major promastigotes. Colocalization of IGF-I and Leishmania was analyzed using immunofluorescence. Following a 24 h in vitro infection, cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, USA), washed in 0.01 M phosphate-buffered saline, pH 7.2 (PBS), blocked for one hour with 2% bovine serum albumin (BSA; Sigma-Aldrich, USA) in PBS, and incubated overnight with monoclonal goat anti-mouse IGF-I antibody (1 : 75; R&D Systems, USA) and a polyclonal mouse anti-Leishmania antibody (1 : 400) [136]. Anti-goat IgG Alexa Fluor-546 (1 : 200, Invitrogen, USA—shown in red) and anti-mouse IgG Alexa Fluor-488 (1 : 400, Invitrogen, USA—shown in green) were used as secondary antibodies. 4,6-Diamidino-2-phenylindole (DAPI, Invitrogen, USA—shown in blue) was used to stain nuclei. Images were captured using a Leica LSM510 confocal microscope with a 63x objective and oil immersion (adapted from Reis et al. [82]).