vvl  (Vector Laboratories)

Journal: PLOS Pathogens

Article Title: Small granule protein CP2 of Cryptosporidium translocates to the parasite-host interface during invasion and localizes to parasitophorous vacuole membrane in association with other secretory proteins

doi: 10.1371/journal.ppat.1013847

Figure Lengend Snippet: (A) Dynamics of CP2-HA secretion during invasion and early growth of C. parvum as revealed by immunofluorescence microscopy. HCT-8 cells were infected with CP2-HA sporozoites, fixed at 15 min, 30 min, 1 h and 2 h post-infection (hpi), and stained with rabbit anti-HA (red), anti- Cp that recognizes whole parasites (green), and Hoechst (blue). Scale bars, 2 μm. (B-E) Immunofluorescence localization of CP2-HA in trophozoites and meronts in HCT-8 cultures. HCT-8 cells were fixed at 24 hpi and stained with rabbit anti-HA (red), Vicia villosa lectin (VVL, green) and Hoechst (blue). Both the top (B and C) and side (D and E) views of the same parasites are shown. Scale bars, 2 μm. (F-G) Ultrastructural localization of CP2-HA in the feeder organelle (F), trophozoite (G), and meront (H) by immunoelectron microscopy of the ileal tissue from mice infected with CP2-HA, using a rabbit anti-HA antibody and 10-nm colloidal gold-conjugated goat-anti-rabbit IgG. Black arrowheads indicate the distribution of gold particles. N, nucleus; SG, small granules. Scale bars, 500 nm.

Article Snippet: After three washes with PBS, the cells were incubated at 37°C for 60 min with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) diluted 1:500 in 1% BSA-PBS, fluorescein-conjugated Vicia villosa lectin (VVL) (Vector; Newark, NJ, USA) diluted 1:500, or fluorescein-conjugated Sporo-Glo polyclonal rat IgG antibody diluted 1:20 (Waterborne, Inc., New Orleans, LA, USA).

Techniques: Immunofluorescence, Microscopy, Infection, Staining, Immuno-Electron Microscopy

Journal: PLOS Pathogens

Article Title: Small granule protein CP2 of Cryptosporidium translocates to the parasite-host interface during invasion and localizes to parasitophorous vacuole membrane in association with other secretory proteins

doi: 10.1371/journal.ppat.1013847

Figure Lengend Snippet: (A-C) Immunofluorescence localization of SG3-HA (cgd6_5400-HA), DG7-HA (cgd6_5420-HA), and DG8-HA (cgd8_5300-HA) in sporozoites. The schematics show the domain structure of SG3, DG7 and DG8, including the putative signal peptide (orange), predicted repeat domains (light blue), and the location of the 3HA tag. Sporozoites from the transgenic lines were fixed and stained with rabbit anti-HA (red), anti-EF1α (green), and Hoechst. Scale bars, 2 μm. (D-F) U-ExM of SG3-HA, DG7-HA and DG8-HA in sporozoites, after expansion in gel and staining with rat anti-HA (green), NHS ester (gey), and Hoechst (blue). Scale bars, 5 μm. (G, I, and K) Immunofluorescence localization of SG3, DG7 and DG8 in trophozoites and meronts in HCT-8 cells at 24 hpi, after staining with rabbit anti-HA (red), Vicia villosa lectin (VVL, green), and Hoechst (blue). Scale bars, 2 μm. (H, J, and L) Co-localization of SG3-HA, DG7-HA and DG8-HA with CP2 in trophozoites and meronts in HCT-8 cells at 24 hpi, after staining with rabbit anti-HA (green), anti-CP2 (red), and Hoechst (blue). Scale bars, 2 μm. (M and N) Ultrastructural localization of SG3-HA and DG8-HA in trophozoites and meronts by immunoelectron microscopy of the ileum from infected mice, using a rabbit anti-HA antibody and a 12-nm colloidal gold-conjugated goat-anti-rabbit IgG antibody. Scale bars, 200 nm.

Article Snippet: After three washes with PBS, the cells were incubated at 37°C for 60 min with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) diluted 1:500 in 1% BSA-PBS, fluorescein-conjugated Vicia villosa lectin (VVL) (Vector; Newark, NJ, USA) diluted 1:500, or fluorescein-conjugated Sporo-Glo polyclonal rat IgG antibody diluted 1:20 (Waterborne, Inc., New Orleans, LA, USA).

Techniques: Immunofluorescence, Transgenic Assay, Staining, Immuno-Electron Microscopy, Infection