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Fig. 4. Immunolocalization of EPMH. (A-P) Methanol/acetone fixed bloodstream cells expressing the GPI¹ EPMH reporter were stained with the indicated primary antibodies and visualized by epifluorescence microscopy. (A-D) Anti-EP₉ and anti-HA. (E-H) Anti-EP₉ and anti-BiP. (I-L) Anti-HA and anti-p67. (M-P) Live trypanosomes were exposed to biotinyl-tomato lectin (TL) at 5°C to allow flagellar pocket binding. After washing, cells were fixed and stained with anti-HA and anti-paraflagellar rod (PFR) antibodies. Paraflagellar rod and tomato-lectin staining were visualized simultaneously in the red channel with appropriate secondary reagents. Three consecutive Z-slices were merged to generate the red channel image. (Q-T) Cells were formaldehyde fixed without permeabilization and stained with anti-HA and anti-EP₉. All panels: left column, merged DIC/DAPI images; right column, merged three channel fluorescent images (arrowheads indicate flagellar pocket localization). DAPI staining (blue) reveals nucleus (n) and kinetoplast (k) localization (indicated in panel A only).

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