Vectashield 19 with dapi

The purified gametocytes from kinesin-8B–GFP, WT-GFP, and Δkinesin-8B parasites were activated in the ookinete medium and then fixed at 0 min, 1–2 min, 6–8 min, and 15 min postactivation with 4% PFA (Sigma-Aldrich) diluted in microtubule stabilising buffer for 10–15 min and added to poly-L-lysine–coated slides. Immunocytochemistry was performed using primary GFP-specific rabbit monoclonal antibody (mAb) (A1122; Invitrogen; used at 1:250) and primary mouse anti-α tubulin mAb (T9026; Sigma; used at 1:1,000). Secondary antibodies were Alexa 488–conjugated antimouse IgG (A11004; Invitrogen) and Alexa 568–conjugated antirabbit IgG (A11034; Invitrogen) (used at 1 in 1,000). The slides were then mounted in Vectashield 19 with DAPI (Vector Labs) for fluorescence microscopy. Parasites were visualised on a Zeiss Axio Imager M2 microscope fitted with an AxioCam ICc1 digital camera (Carl Zeiss, Inc).
To measure nuclear DNA content of activated microgametocytes by direct immunofluorescence, images of parasites fixed (0 mpa and 8–10 mpa) and stained as above were analysed using the ImageJ software (version 1.44) (National Institute of Health) as previously described (Tewari et al, 2005 (link)).

The Pbkinesin-8X-GFP gametocytes were purified and activated in ookinete medium then fixed at 0 min, 1–2 min, 6–8 min and 15 min post-activation with 4% paraformaldehyde (PFA, Sigma) diluted in microtubule stabilising buffer (MTSB) for 10–15 min and added to poly-L-lysine coated slides. Immunocytochemistry was performed using primary GFP-specific rabbit monoclonal antibody (mAb) (Invitrogen-A1122; used at 1:250) and primary mouse anti-α tubulin mAb (Sigma-T9026; used at 1:1000). Secondary antibodies were Alexa 488 conjugated anti-mouse IgG (Invitrogen-A11004) and Alexa 568 conjugated anti-rabbit IgG (Invitrogen-A11034) (used at 1 in 1000). The slides were then mounted in Vectashield 19 with DAPI (Vector Labs) for fluorescence microscopy. Parasites were visualised on a Zeiss AxioImager M2 microscope fitted with an AxioCam ICc1 digital camera (Carl Zeiss, Inc).

The purified schizonts from PbKinesin-5-GFP parasites were fixed in 2% paraformaldehyde (PFA) and smeared on poly-L-lysine coated slides. Purified gametocytes were activated in ookinete medium then fixed at 1, 2, 4, 6, 8, and 15 min post-activation with 4% PFA diluted in microtubule stabilizing buffer (MTSB) for 10–15 min and added to poly-L-lysine coated eight-well slides. Immunocytochemistry was performed using primary GFP-specific rabbit monoclonal antibody (mAb) (Invitrogen-A1122; used at 1:250) and primary mouse anti-α tubulin mAb (Sigma-T9026; used at 1:1000) or mouse anti-centrin mAb (Millipore-04-1624, used at 1:500) or anti-NDC80 (polyclonal sera, a kind gift from Marc-Jan Gubbels). Secondary antibodies were Alexa 488 conjugated anti-mouse IgG (Invitrogen-A11004) and Alexa 568 conjugated anti-rabbit IgG (Invitrogen-A11034) (used at 1 in 1000). The slides were then mounted in Vectashield 19 with DAPI (Vector Labs) for fluorescence microscopy. Parasites were visualized on a Zeiss AxioImager M2 microscope. Pearson’s colocalization coefficient (R) was calculated using image J software (version 1.44).