Axio imager m2 microscope

Blood containing approximately 50,000 parasites of the kinesin-8X-KO line was injected intraperitoneally (i.p.) into mice to initiate infections. Asexual stages and gametocyte production were monitored by microscopy on Giemsa stained thin smears. Four to five days post-infection, exflagellation and ookinete conversion were examined as described previously [70 (link)] with a Zeiss AxioImager M2 microscope (Carl Zeiss, Inc) fitted with an AxioCam ICc1 digital camera. To analyse mosquito transmission, 30–50 Anopheles stephensi SD 500 mosquitoes were allowed to feed for 20 min on anaesthetized, infected mice whose asexual parasitaemia had reached 15% and were carrying comparable numbers of gametocytes as determined on Giemsa stained blood films. To assess mid-gut infection, approximately 15 guts were dissected from mosquitoes on day 14 post-feeding and oocysts were counted on an AxioCam ICc1 digital camera fitted to a Zeiss AxioImager M2 microscope using a 63x oil immersion objective. On day 21 post-feeding, another 20 mosquitoes were dissected, and their guts and salivary glands crushed separately in a loosely fitting homogenizer to release sporozoites, which were then quantified using a haemocytometer or used for imaging. Mosquito bite back experiments were performed 21 days post-feeding using naive mice and blood smears were examined after 3–4 days.

Blood containing approximately 50,000 parasites of the Δkinesin-8B line was injected intraperitoneally (i.p.) into mice to initiate infections. Asexual stages and gametocyte production were monitored by microscopy on Giemsa-stained thin smears. 4–5 d postinfection, exflagellation and ookinete conversion were examined as described previously (Guttery et al, 2012 (link)) with a Zeiss Axio Imager M2 microscope (Carl Zeiss, Inc) fitted with an AxioCam ICc1 digital camera. To analyse mosquito transmission, 30–50 Anopheles stephensi SD 500 mosquitoes were allowed to feed for 20 min on anaesthetized, infected mice with an asexual parasitemia of 15% and a comparable number of gametocytes as determined on Giemsa-stained blood films. To assess midgut infection, ∼15 guts were dissected from mosquitoes on day 14 postfeeding, and oocysts were counted on an AxioCam ICc1 digital camera fitted to a Zeiss Axio Imager M2 microscope using a 63× oil immersion objective. On day 21 postfeeding, another 20 mosquitoes were dissected, and their guts crushed in a loosely fitting homogenizer to release sporozoites, which were then quantified using a haemocytometer or used for imaging. Mosquito bite back experiments were performed 21 d postfeeding using naive mice, and blood smears were examined after 3–4 d.

Blood containing ∼50,000 parasites of the ΔSAS44 line was injected i.p. into mice to initiate infections. Asexual stages and gametocyte production were monitored by microscopy on Giemsa-stained thin smears. 4–5 d post-infection, exflagellation and ookinete conversion were examined as described previously (Zeeshan et al, 2019b (link)) with a Zeiss AxioImager M2 microscope (Carl Zeiss, Inc.) fitted with an AxioCam ICc1 digital camera. To analyse mosquito transmission, 30–50 Anopheles stephensi SD 500 mosquitoes were allowed to feed for 20 min on anaesthetized, infected mice with an asexual parasitaemia of 15% and a comparable number of gametocytes as determined on Giemsa-stained blood films. To assess midgut infection, ∼15 guts were dissected from mosquitoes on day 14 post-feeding, and oocysts were counted on an AxioCam ICc1 digital camera fitted to a Zeiss AxioImager M2 microscope using a 63× oil immersion objective. On day 21 post-feeding, another 20 mosquitoes were dissected, and their guts crushed in a loosely fitting homogenizer to release sporozoites, which were then quantified using a haemocytometer. Mosquito bite back experiments were performed 21 d post-feeding using naive mice, and blood smears were examined after 3–4 d.

Worms tagged with fluorescence were imaged under DIC and fluorescence using an Axio Imager M2 microscope (ZEISS). Images were processed and viewed using ZEN 2 pro software (ZEISS). An Immersol 518F oil (Zeiss) was used. All the images were captured at 20°C (Gan et al., 2019 (link)).
Time-lapse imaging of HIS-24::mCherry in N2 and trim-21 mutants was performed at 20°C under a ×100 oil objective using an Axio Imager M2 microscope (ZEISS). Images were focused on specific corpses and were taken every few minutes until the corpses disappeared.

Sporozoites were isolated from midgut and salivary glands from mosquitoes infected with WT‐GFP and ∆MyoE parasites between Days 21 and 24 post infection. Isolated sporozoites in RPMI 1640 containing 3% bovine serum albumin (Fisher Scientific) were pelleted (5 min, 5,000 rpm, 4°C). The motility assay was performed as described previously (Moreau et al., 2017). Briefly, a drop (6 μl) of sporozoites was transferred onto a microscope glass slide with a cover slip. Time‐lapse videos of sporozoites (one frame every 1 s for 100 cycles) were taken using the differential interference contrast settings with a 63× objective lens on a Zeiss AxioImager M2 microscope fitted with an AxioCam ICc1 digital camera and analysed with the AxioVision 4.8.2 software. The assay using Matrigel was performed as described previously (Volkmann et al., 2012). A small volume (20 μl) of sporozoites, isolated as above for WT‐GFP and ∆MyoE parasites, were mixed with Matrigel (Corning). The mixture (6 μl) was transferred on a microscope slide with a cover slip and sealed with nail polish. After identifying a field containing sporozoites, time‐lapse videos (one frame every 2 s for 100 cycles) were taken using the differential interference contrast settings with a 63× objective lens on a Zeiss AxioImager M2 microscope fitted with an AxioCam ICc1 digital camera and analysed with the AxioVision 4.8.2 software.

Specific antigen binding was determined by incubating sections with the respective primary antibodies for Cx43 (Cell Signalling Technology, Cambridge, UK), C3aR (Bioss, Woburn, MA, USA), and IL-1β (Abcam, Cambridge, UK) overnight at 4°C. A biotinylated IgG (Life Technologies, Carlsbad, CA, USA) was used as secondary antibody. Signal amplification was performed by using the VECTASTAIN® ABC Kit (Vector Laboratories, Burlingame, CA, USA), and signal was obtained by developing sections with the VECTOR® NovaRED™ Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA, USA). Cell nuclei were counterstained in haematoxylin. Sections were investigated by bright-field microscopy using an Axio Imager M.2 microscope and the Zeiss ZEN 2.3 software (Zeiss, Jena, Germany). Results are presented as mean pixel density. PAS staining was performed using a PAS-staining kit (Merck Millipore, Darmstadt, Germany). Signal density was measured using an Axio Imager M.2 microscope and the Zeiss ZEN 2.3 software (Zeiss, Jena, Germany). Results are presented as mean density.

Different developmental stages of parasites during schizogony, gametogenesis and zygote to ookinete transformation were analyzed for SAS4-GFP expression and localization using a 63× oil immersion objective on a Zeiss AxioImager M2 microscope. Purified male gametocytes were examined for GFP expression and localization at different time points (1–15 min) after activation in ookinete medium containing xanthurenic acid. Images were captured using a 63× oil immersion objective on a Zeiss AxioImager M2 microscope fitted with an AxioCam ICc1 digital camera (Carl Zeiss, Inc.). Time-lapse videos (1 frame every 5 s for 10 cycles) were taken with a 63× objective lens on the same microscope and analyzed with the AxioVision 4.8.2 software as described recently (Zeeshan et al, 2020b (link)).