Openlab 5

For the compound microscopy data shown in Fig 4, images were taken using a Zeiss Axioskop with Hamamatsu CCD or ORCA cMOS camera equipped with 63x 1.4NA Plan Apochromat oil immersion objective. Carl Zeiss filter sets 49, 38, and 43HE were used for the visualization of DAPI, Alexa 488, and Alexa 555 respectively. An X-Cite 120Q lamp (Lumen Dynamics) was used as the fluorescence light source. Openlab 5.5.2 (PerkinElmer) and Micromanager [101 (link), 102 (link)] were used as acquisition software. For all other figures, a Leica TCS SP8 confocal microscope driven by LAS software version 3.3.1 or X was used. This laser scanning confocal microscope was equipped with Photomultiplier (PMT) and Hybrid detectors (HyD). For all images, a 63x 1.4NA HC Plan Apochromat oil immersion objective was used with 100–200% zoom for immunostaining, and 300% zoom for single molecule FISH, using the standard scanner with 400Hz scanning speed. For figure preparation, contrast was linearly adjusted in Adobe Photoshop identically across all samples. In some cases, images were merged using the stitching plugin in FIJI/Image J [103 (link)] to generate whole gonad images.

The assay was performed as described [11 (link)] with minor modifications. DG627, JK5233, JK5235 animals were raised at 15°C until 36 hours past mid-L4, then moved to plates pre-incubated at 25°C and maintained at 25°C for 12.5 hours. We chose 12.5 hours because germ cell counts became unreliable with longer times (nuclear morphology became increasingly compromised after incubations of 13 hours and longer). Next, gonads were dissected, fixed, and stained for anti-PH3, anti-GLD-1 and DAPI (see staining section below). To estimate the number of cells within the distal pool, we manually counted the number of M-phase arrested germ cells distal to the GLD-1 boundary (as assessed by DAPI morphology and PH3 staining) using the cell counter tab in Openlab 5.5.2 (PerkinElmer). Scoring was done blind to genotype. We excluded samples with abnormal, fragmented nuclei that made cell counting unreliable (22–49% per genotype). We note that not every nucleus distal to the GLD-1 boundary was arrested in M-phase in some gonads but these few nuclei were included in the “distal pool” counts.

Endocytic uptake of the fluorescent dye “dextran Alexa Fluor 594” (Life Technologies, cat. D22913) by PV organelles was achieved as described previously [47 (link)]. Briefly, trophozoites were grown and harvested as described above. Cells were washed twice in 1x PBS (900 x g, 10 min, 4°C) and resuspended in PBS supplemented with 5 mM cysteine, 5 mM glucose, and 0.1 mM ascorbic acid containing 4 mg/ml dextran Alexa Fluor 594. Trophozoites were transferred to 37°C for 30 minutes, protected from light. Samples were then washed twice in PBS (900 x g, 10 min, 4°C), fixed in 3% formaldehyde and processed for immunofluorescence [48 (link)]. For immunofluorescence staining, a fluorescein isothiocyanate (FITC)-conjugated mouse anti-HA antibody (dilution 1:100; Roche Diagnostics GmbH, Manheim, Germany) was used. Prior to inspection, specimens were embedded in Vectashield (Vector Labs, Inc, cat. H-1200) containing the DNA intercalating agent 49-6-Diamidino-2-phenylindole (DAPI). Immunofluorescence analysis was performed on a Nikon Eclipse 80i microscope using the software Openlab 5.5.2 (PerkinElmer) for picture acquisition.

Staining was performed using 4-μm sections following the protocol described in (Huang et al., 2010 (link)). The frequency of beta cell containing P62 and polyubiquitinated proteins inclusions were assessed in the adjacent sections co-stained for P62 and insulin or K63 polyubiquitin and insulin, and were mounted with Vectashield with DAPI (Vector Laboratories cat. No. H-1200, RRID:AB 2336790). All islets on the section were viewed using a Leica DM6000 fluorescent microscope with a ×20 objective and imaged using OpenLab 5.5 software (Improvision). Six to thirty islets per case were imaged; the number of insulin positive beta cells counted were 1,626 and 1,498 per control and hIAPP ASO treatment in 1 mg/kg groups, and counted being 2,187 and 6,838 in 5 mg/kg groups respectively. Image analysis was performed blindly by two independent investigators (TG and ZW). The frequency of beta cells with impaired autophagosome/lysosome mediated protein degradation was expressed as percentage of insulin positive cells containing P62 inclusions.

Subcutaneously located ticks were removed with the skin and the surrounding tissue from the complete fur and immediately fixed in 10% buffered formalin, dehydrated in ascending ethanol series (20–100%) and embedded in paraffin blocks. Seven-micron thick sections were cut with a rotary microtome (Leica RM 2155, Leica Microsystems Nussloch GmbH, Nussloch, Germany), placed on glass slides and stained with hematoxylin and eosin (H&E). Microscopy and pictures were performed using a Zeiss Axioplan 2 imaging (Carl Zeiss Microscopy, Jena, Germany) equipped with an objective 25×/0.8 Plan-Neofluar Oil and a color camera ProgRes C14 (Jenoptik, Jena, Germany). Whole section overviews were obtained by tile scans with a 25×/0.8 Plan-Neofluar Oil objective using a tile scan routine under Openlab 5.5 (Improvision, Coventry, UK). During histological examination it was noticed that the inflammatory response was related to the stage of degradation of the tick. Therefore, the ticks were divided into 3 categories: (i) well-preserved ticks with correct position of hypostome and the appendages and intact exoskeleton and body; (ii) deformed ticks with intact exoskeleton; and (iii) ticks with broken, fully deformed exoskeleton.