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. 2018 Feb 7;9:130. doi: 10.3389/fimmu.2018.00130

Figure 9.

Figure 9

Gbp2b/Gbp1 protein and Leishmania major parasites in skin of infected mice. Slices of skin tissue of females of BALB/c (A), STS (B), CcS-5 (C), CcS-20 (D), and O20 (E) mice infected for 8 weeks with L. major were stained with the anti-Leishmania lipophosphoglycan mouse monoclonal antibody (cat. no. CLP003A, Cedarlane, Hornby, Canada) and TRITC labeled IgM (115-025-020, Jackson ImmunoResearch, West Grove, PA) all diluted 1:500 and the rabbit anti-Gbp1 Polyclonal antibody (PA5-23509, Thermo Fisher Scientific, Rockford, IL, USA) diluted 1:100 and anti-rabbit-AlexaFluor-647 (cat. no. 711-605-152; Jackson ImmunoResearch, West Grove, PA) diluted 1:500. Nuclei of the cells were stained with bisBenzimide H33258 (Sigma-Aldrich, St. Louis, MO, USA) 10 mg per 1 ml diluted 1:1,000. Images were captured with microscope Leica DM6000 objective HCX PL Apo 40×/0.75 PH2 and color camera Leica DFC490. Evaluation of images was done with Fiji ImageJ 1.51n. Figures are representatives of data from 8 to 11 mice (see Materials and Methods) in 3 of them 10 fields (320.66 × 239.57 µM) from each mouse were analyzed, in the rest one field was analyzed to verify the results. White arrow shows L. major amastigotes (red color), green arrows show Gbp2b/Gbp1 protein (yellow color), red arrows point to amastigotes co-localized with Gbp2b/Gbp1, whereas yellow arrows show either Gbp2b/Gbp1 surrounding parasite clusters or stretch of parasites and Gbp2b/Gbp1. Cell nuclei are stained in blue.