Fig. 6

HCAb2 localized specifically to MDA-MB-231 xenograft tumors in immunodeficient mice. a-e Representative images showing localization of HCAb2 to tumors at 2 h, 6 h and 24 h time points. Female NSG mice bearing tumors ranging from 300-500 mm³ were retro-orbitally injected with 12 μg of HCAb1 (n = 2) and HCAb2 (n = 3) into respective animals. After 2 h, 6 h and 24 h mice were euthanized and tumors along with various normal tissues were stained to detect HCAb1 and HCAb2 localization. HCAb1 did not localize to the tumors (panels a and b), while HCAb2 localized to tumors at the earliest time point (panel c) and the later time points of 6 h (panel d) and 24 h (panel e). Arrows indicate cells with HCAb2 staining. Insets reveal magnified image and scale bar represents 20 μm. f-j Immunofluorescence analysis of mouse normal tissues to determine distribution of HCAb2. Frozen sections of brain (panel f), heart (panel g), lung (panel h), liver (panel i) and kidney (panel j) tissues from 24 h time point mouse were analyzed to detect the presence of HCAb2. Low levels of HCAb2 were detected in heart (panel g), liver (panel i) and kidney tissues (panel j). Arrows indicate HCAb2 localization. Insets reveal magnified image and scale bar represents 20 μm. k-n Immunofluorescence analysis of xenograft tumor tissue to identify HCAb2 localization in MDA-MB-231 cells. 24 h time point tumor section was incubated with anti-CD44-FITC and Alexa Fluor® 594 anti-mouse IgG antibodies. From panels l, m and n, it can be observed that HCAb2 localizes to MDA-MB-231 cells. Arrows indicate cells with positive CD44 and HCAb2 staining. Scale bar represents 10 μm