protein expression  (Addgene inc)

Journal: International journal of molecular sciences

Article Title: Host-Parasite Interactions in Toxoplasma gondii -Infected Cells: Roles of Mitochondria, Microtubules, and the Parasitophorous Vacuole.

doi: 10.3390/ijms252413459

Figure Lengend Snippet: Figure 9. Confirmation of the specificity of the EB1 antibody. HFF cells were infected with Tg RH as described before, and 24 h later, the cells were fixed and examined by secondary (indirect) immunofluorescence microscopy. (Panel A) = Overlay image of nuclear (N) DNA in blue (DAPI), EB1 in red (anti-EB1 primary antibody, Alexafluor 594 secondary antibody), and microtubules in green (anti-tubulin primary antibody, Alexafluor 488 secondary antibody). (Panel B) = Red (EB1) only. Due to the high conservation of tubulin sequences through phylogeny, the antibody to human tubulin cross-reacts with T. gondii tubulin, which is visible in the parasite subpellicular and spindle regions, as evidenced by the strong green color of the parasite cells inside the PV.

Article Snippet: The plus-end-associated protein EB1 was visualized using a rabbit polyclonal antibody raised against human EB1 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Infection, Immunofluorescence, Microscopy

Journal: International journal of molecular sciences

Article Title: Host-Parasite Interactions in Toxoplasma gondii -Infected Cells: Roles of Mitochondria, Microtubules, and the Parasitophorous Vacuole.

doi: 10.3390/ijms252413459

Figure Lengend Snippet: Figure 10. Depolymerization and recovery of host microtubules reveal nucleation of novel micro- tubules at or near the PVM. Human foreskin fibroblasts (HFF) were infected with RH T. gondii. Twenty-four hours after infection, HFF cells were placed at 4 ◦C for 45 min to depolymerize mi- crotubules and then placed at room temperature and fixed in methanol at intervals. (Panels A,B) show an infected cell fixed immediately after cold treatment, demonstrating depolymerization of microtubules stained with anti-tubulin (green) and anti-EB1 (red) antibodies, and DNA stained by DAPI (blue). (Panel A) shows a merged image of all three channels, while (B) shows tubulin alone. The arrow indicates cold-resistant microtubule staining in Tg tachyzoites. To focus exclusively on host microtubules, cells in (panels C,D) were assayed by immunofluorescence microscopy against human EB1 (green), and co-stained with DAPI (blue). (Panels C,D) are representative images of infected cells at 4 min post-release from 4 ◦C, and the arrows indicate nascent human microtubules forming at or near the PVM. The positive staining located near the host nucleus represents the outgrowth of EB1-bound microtubules from the microtubule organizing center (MTOC).

Article Snippet: The plus-end-associated protein EB1 was visualized using a rabbit polyclonal antibody raised against human EB1 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Infection, Staining, Immunofluorescence, Microscopy