Ultrastructural Imaging of Plasmodium Gametocytes

Purified gametocytes were activated for 1–2 min and then activation was stopped by adding 4% formaldehyde. Sample preparation of P. berghei parasites for UExM was performed as previously described^{68 (link),69 (link)}, except that 4% formaldehyde (FA) was used as fixative^{43 (link)}. Fixed samples were then attached on a 12 mm round poly-d-lysine (A3890401, Gibco) coated coverslip for 10 min. Immuno-labelling was performed using primary antibodies against α-tubulin and β-tubulin (1:200 dilution, AA344 and AA345 from the Geneva antibody facility), anti-γ-tubulin antibody (1:500 dilution, Sigma T5192) and anti-HA antibody (3F10) (1:250 dilution, Roche). Secondary antibodies anti-guinea pig Alexa 647, anti-rabbit Alexa 405 and anti-rat Alexa 488 were used at dilutions 1:400 (Invitrogen). Atto 594 NHS-ester was used for bulk proteome labelling (Merck 08741). 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.