Alexa 568 conjugated anti rabbit igg

HeLa cells were cultured in DMEM containing 10% FBS in a humidified atmosphere containing 5% CO₂. After treatment with each compound for 2.5 h (interphase MT network) or 5 h (mitotic spindle), the cells were immediately fixed with cold MeOH (−20 °C). After blocking with PBS containing 0.5% bovine serum albumin, cells were incubated with anti-α-tubulin antibodies (1:500 dilution, Santa Cruz, Cat# sc-32293) to observe interphase cells, or with anti-BubR1 (1:250 dilution, BD Bioscience, Cat# 612502) and anti-α-tubulin (1:250 dilution, MBL, Cat# PM054) antibodies to observe mitotic spindle. After staining with Alexa⁴⁸⁸-conjugated anti-mouse IgG (1:2,000 dilution, Invitrogen, Cat#A11001) and Alexa568-conjugated anti-rabbit IgG (1:2000 dilution, Invitrogen, Cat#A11011), cells were washed four times with PBS and mounted with PBS containing 0.1 μg ml⁻¹ DAPI. MTs and BubR1 signals were observed under a Leica LAS AF 6000 fluorescence microscope (Leica Microsystems) or an LSM 700 laser-scanning confocal microscope (Zeiss).

The kinesin-GFP gametocytes were purified, activated in ookinete medium, fixed with 4% paraformaldehyde (PFA, Sigma) diluted in MT stabilising buffer (MTSB) for 10 to 15 min and added to poly-L-lysine coated slides. Immunocytochemistry was performed using primary GFP-specific rabbit mAb (Invitrogen-A1122; used at 1:250) and primary mouse anti-α tubulin mAb (Sigma-T9026; used at 1:1,000). Secondary antibodies were Alexa 488 conjugated anti-mouse IgG (Invitrogen-A11004) and Alexa 568–conjugated anti-rabbit IgG (Invitrogen-A11034) (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 AxioImager M2 microscope fitted with an AxioCam ICc1 digital camera.

The ARK2-GFP gametocytes were purified, activated in ookinete medium, fixed at different time points with 4% paraformaldehyde (PFA, Sigma) diluted in MT-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. Post-acquisition analysis was carried out using Icy software – version 1.9.10.0. Images presented are 2D projections of deconvoluted Z-stacks of 0.3 μm optical sections.

The purified SAS4-GFP gametocytes were activated in ookinete medium then fixed at 2–8 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) (A1122; used at 1:250; Invitrogen) and primary mouse anti–α tubulin mAb (T9026; used at 1:1,000; Sigma-Aldrich). Secondary antibodies were Alexa 488–conjugated anti-mouse IgG (A11004; Invitrogen) and Alexa 568–conjugated anti-rabbit IgG (A11034; Invitrogen) (used at 1 in 1,000). The slides were then mounted in VECTASHIELD 19 with DAPI (Vector Labs) for fluorescence microscopy.

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).