Localization of Babesia microti P53 Protein

The cellular localization of BmP53 was determined by an Immunofluorescence antibody assay (IFA) according to [14 (link)]. Briefly, thin blood smears of B. microti-infected erythrocytes or non-infected isolated hamster platelets were fixed in a solution of 95% methanol and 5% acetone (v:v) at -20°C for 30 min and then probed with mouse anti-rGST-BmP53tr1-TSP1 immune sera diluted in 4% fetal bovine serum in PBS (1:100) for 1 h at 37°C in a moist chamber. A secondary antibody of goat anti-mouse or -Rabbit IgG (H+L) (Alexa-Fluor 488 conjugate) (green dye) and goat anti-mouse IgG (H+L) (Alexa-Fluor 594-conjugate) (red dye) (Life Technologies, USA) diluted in the same buffer (1:250) was applied on the smears and then incubated for 1 h at 37°C. DNA was stained with Hoechst blue dye (Dojindo Molecular Technologies, Inc., USA) in PBS (1:250) for 10 min at RT. After washing with PBS-T, the smears were mounted using a fluorescent mounting medium (Dako, USA) and then covered with a glass coverslip. Imaging was performed using All-in-One Fluorescence Microscope, BZ-X700 series (KEYENCE corporation, Japan). For the co-localization study, anti-rBmSA1 rabbit serum [43 (link)] was used as a specific marker for B. microti Gray strain parasites.