Ultrastructural Imaging of Activated Gametocytes and Ookinetes

Purified gametocytes were activated for 4 to 5 min and 15 min; activation was stopped by adding 1X ice-cold PBS. Activated gametocytes and mature ookinetes were sedimented onto 12-mm round Poly-D-Lysine (A3890401, Gibco) coated coverslips for 10 min (gametocyte procedure was performed on ice), fixed in methanol at −20°C for 7 min, and then prepared for U-ExM as described previously [45 (link),74 (link)]. Immuno-labelling was performed using primary antibody against α-tubulin and β-tubulin (1:200 dilution, source: Geneva antibody facility) and secondary antibody anti-guinea pig Alexa 488 (1:400 dilution, source: ThermoFisher). Images were acquired on a Leica TCS SP8 microscope; image analysis was performed using Fiji-Image J and Leica Application Suite X (LAS X) software.

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Zeeshan M., Rashpa R., Ferguson D.J., Abel S., Chahine Z., Brady D., Vaughan S., Moores C.A., Le Roch K.G., Brochet M., Holder A.A, & Tewari R. (2022). Genome-wide functional analysis reveals key roles for kinesins in the mammalian and mosquito stages of the malaria parasite life cycle. PLoS Biology, 20(7), e3001704.

Publication 2022

Poly-D-Lysine TCS SP8 microscope Fiji-Image J Leica Application Suite X (LAS X)

Antibody Cold Dilution Guinea pig Lysine Methanol Microscope Poly Tubulin α tubulin

Corresponding Organization : University of Nottingham

Other organizations : University of Geneva, John Radcliffe Hospital, University of Oxford, Science Oxford, Oxford Brookes University, University of California, Riverside, Birkbeck, University of London, Institute of Structural and Molecular Biology, The Francis Crick Institute

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Variable analysis

independent variables

Activation time of gametocytes (4 to 5 min and 15 min)

dependent variables

Morphology and characteristics of activated gametocytes and mature ookinetes
Immunolabeling patterns of α-tubulin and β-tubulin

control variables

Fixation method (methanol at -20°C for 7 min)
Coating of coverslips (Poly-D-Lysine)
Sedimentation time of activated gametocytes and mature ookinetes (10 min)
Primary antibody (α-tubulin and β-tubulin, 1:200 dilution)
Secondary antibody (anti-guinea pig Alexa 488, 1:400 dilution)
Microscope (Leica TCS SP8)
Image analysis software (Fiji-Image J and Leica Application Suite X (LAS X))

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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