File:Indirect-immunofluorescence-assay-IFA-of-a-diprotist-culture-containing-Colpodella-sp.png

English: Indirect immunofluorescence assay (IFA) of a diprotist culture containing Colpodella sp. ATCC 50594 and Parabodo caudatus (homotypic synonym: Bodo caudatus, according to NCBI Taxonomy) and IFA of Plasmodium falciparum schizonts. Antiserum 686 specific for the high molecular weight P. falciparum rhoptry protein RhopH3 and anti-β-tubulin monoclonal antibody KMX-1 of Physarum polycephalum. Samples of the diprotist culture containing Colpodella sp. ATCC 50594 and Parabodo caudatus were fixed with absolute methanol. Panels A-D. Diprotist culture was incubated with antiserum 686 (1:100; green) and anti-β-tubulin KMX-1 (1:1000; red) followed by incubation with ALEXA-488 Goat anti-rabbit antibody (1:1000) and ALEXA 633 Goat anti-mouse antibody (1:1000). Smears were mounted with Vectashield containing DAPI for nuclear staining. Panel A, Merge of antiserum 686, KMX-1 and DAPI showing 686 antibody reactivity with cytoplasmic vesicles in the anterior section of Colpodella trophozoites and the apical tip of the cell; B, DAPI nuclear staining, C, KMX-1 reactivity on cell body and flagella of Colpodella trophozoite (red) and D, antiserum 686 reactivity (green) showing protein recognition and reactivity of the cytoplasm and vesicles. Panels E-H. No RhopH3 antibody reactivity was observed with Parabodo caudatus cells. Merge of antiserum 686, KMX-1 and DAPI (E) showing DAPI and anti-β-tubulin staining with no 686 antibody reactivity. Panel F shows DAPI staining of nucleus and kinetoplast of Parabodo caudatus. Panel C shows reactivity of anti-β-tubulin antibody KMX-1 with the cell body of Parabodo caudatus with no flagella staining. Panel H shows no reactivity of antiserum 686 with Parabodo caudatus. Panels I-L. Diprotist culture was incubated with normal rabbit (1:100) and normal mouse serum (1:200) followed by incubation with ALEXA-488 Goat anti-rabbit antibody (1:1000) and ALEXA 633 Goat anti-mouse antibody (1:1000). Smears were mounted with Vectashield containing DAPI for nuclear staining. I, merge showing DAPI staining and no protein reactivity, J, DAPI alone, K & L, no protein reactivity on Colpodella trophozoites or Parabodo caudatus with normal rabbit and mouse serum. Panels M-P. Segmented P. falciparum (strain 3D7) schizonts fixed in ice-cold methanol and acetone (1:1 ratio) were incubated with antiserum 686 (1:100; green) and KMX-1 (red). Merge of DAPI, RhopH3 and β-tubulin showing antibody reactivity with merozoite rhoptries showing punctate staining of rhoptries (M), DAPI nuclear staining (N), β-tubulin (O) and antiserum 686 reactivity alone (P).