Axio imager z2 fluorescent microscope

Paraffin embedded sALS patient postmortem motor and occipital cortex tissue sections (see Supplemental File 5) were deparaffinized, immunostained, and imaged as previously described ^{30 (link),31 (link)}. Primary antibodies used were rabbit anti-TDP-43 (ProteinTech 10782-2-AP) and guinea pig anti-Map2 (Synaptic Systems 188004). Goat anti-rabbit Alexa 488 and goat anti-guinea pig Alexa 568 (Thermo Fisher Scientific) were used as secondary antibodies. Tissue sections were imaged with a 20X objective on a Zeiss Axioimager Z2 fluorescent microscope housing an apotome2 module. All images were acquired with identical exposure times. Categorical analysis was carried about by manual observation and counting of cells displaying strong nuclear TDP-43 immunoreactivity, strong nuclear with cytoplasmic TDP-43 immunoreactivity, weak nuclear TDP-43 immunoreactivity, nuclei completely devoid of TDP-43 immunoreactivity, and cells with visible cytoplasmic TDP-43 aggregates. Nuclear/cytoplasmic ratios of TDP-43 were quantified as previously described ^{31 (link)} whereby integrated density was measured within a nuclear ROI and cytoplasmic ROI within the cell body. Background mean intensity values were subtracted prior to calculating nuclear/cytoplasmic ratios.

Analysis of ALK, ROS1, and MET by FISH was conducted using Vysis^® LSI ALK Dual Color Break Apart FISH Probe, ZytoLight^® ROS1 SPEC Dual Color Break Apart, and ZytoLight^® MET/CEN7 Dual Color Probe, respectively. The Zeiss Axio Imager Z2 fluorescent microscope, ×100 objective, and Isis software were used for scoring each case and image acquisition.
Reading was carried out in double filter or alternating red and green filters by two experienced molecular biologists trained in interpretation and applying the interpretative guidelines provided by the manufacturer and the published recommendations 30, 31. A hundred nuclei for each CMA and fifty nuclei for each TMA were counted. FISH locus rearrangements were considered as positive in relation to two different patterns: the classic break‐apart pattern with one fusion signal and two separated orange and green signals (more than two signal diameters apart) and the atypical pattern with a single red (in case of ALK) or green (in case of ROS1) signal in addition to fused signals. FISH‐positive cases for both ALK and ROS1 rearrangements were defined as more than 15% split or single signals. Concerning amplification, an average copy number of ALK or MET inferior to 6 was considered negative 32.

The fluorescent in situ assay shown in Figures 5C, 5E, 6I, S4B, S4C, and S7G–S7I was performed using the RNAscope 2.5 HD—multiplex fluorescent Manual Assay kit (ACDbio Cat. 320850). In this assay tissue was prepared and pretreated in the same way as in the chromogenic assay (see above). After incubation with target probes, 3 amplification steps and 1 detection step were performed following manufacturer instructions. Slides were mounted using the SlowFade Gold with DAPI mounting media (Life Tech Cat. S36939) covered with 1.5 glass coverslip (Fisher Cat. 12544E) and sealed with clear nail polish. Slides were imaged using the Axio Imager.Z2 fluorescent microscope (Zeiss). A 16 bit 1388 X 1040 pixel images were acquired using AxioCam HR3 camera (Zeiss). Fluorescent signal was imaged at 20x magnification using the Apotome.2 module (Zeiss). Z stack images (optical slice 1 μm) were obtained for each image. Set exposure was used when comparing signal between WT and KO mice for a given experiment. For cell type specific expression quantification experiments shown in Figures 5C–5E slides were imaged using the Zeiss LSM 710 confocal microscope at 20X magnification. 16 bit images at 0.16 μm/pixel were generated. A z stack (optical slice 1 μm, 6 slices total) was obtained for each image. Example images show maximal intensity projections for each group.

Non-neurological control and C9orf72 patient postmortem paraffin embedded motor cortex sections were obtained from the Target ALS Human Postmortem Tissue Core (see Additional file 5: Supplemental Table 2 for demographic information). Antigen retrieval and immunofluorescent staining was conducted as previously described [3 (link)]. Antibodies for immunostaining are as follows: 1:500 Rabbit Anti-Lamin B1 (Abcam ab16048), 1:1000 Guinea Pig Anti-Map2 (Synaptic Systems 188,004), 1:1000 Goat Anti-Guinea Pig Alexa 488 (Invitrogen A11073), 1:1000 Goat Anti-Rabbit Alexa 647 (Invitrogen A21245). Nuclei from Map2 positive Layer V neurons were imaged with a 63X objective and a Zeiss Axioimager Z2 fluorescent microscope equipped with an apotome2 module. All images were acquired using identical exposure times. Images are presented as default apotome processed images generated in Zeiss Zen Blue 2.3.

Thin smears of parasite-infected blood, spots of purified sporozoites, and oocyst-attached mosquito midguts were prepared on glass slides, stained with Hoechst 33342, and fluorescent and differential interference contrast (DIC) images were captured using an AxioCam MRm CCD camera (Carl Zeiss, Germany) fixed to an Axio imager Z2 fluorescent microscope with a Plan-Apochromat 100 ×/1.4 oil immersion lens (Carl Zeiss) and Axiovision software (Carl Zeiss). Obtained images were processed using Zen lite 2011 software (Carl Zeiss) and Adobe Photoshop CS (Adobe Systems Inc., San José, CA).
The segregation efficiency of the plasmid per nuclear division was calculated using the following equation:
$\mathrm{s}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{e}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y}=100\times {(\mathrm{P}\mathrm{g}\mathrm{f}\mathrm{p}/100)}^{1/n}$
, where P_gfp is the percentage of GFP-positive parasites after PYR removal and n is the number of nuclear divisions. In this study, we assumed that 3.5 rounds of nuclear division (equivalent to the generation of 11 daughter parasites) occurred during one blood-stage schizogony of 18 hours.