Axio imager z1

All images and analyses were generated by personnel who had no knowledge of the mouse genotype. Aβ images were obtained using a Zeiss Axio Imager Z1 fluorescence microscope (Carl Zeiss Microscopy, Jena, Germany) with a 10× lens objective. Mosaic images of the entire cortex and hippocampus of each animal were obtained and analyzed, and plaque burden was calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA). This was done by isolating the cortex or hippocampus, thresholding to a standard value and calculating the area occupied. Using an UltraVIEW VoX spinning disc confocal microscope (PerkinElmer, Waltham, MA, USA), hippocampal PSD-95 immunoreactive puncta were imaged with a 60× lens objective and digitally magnified to × 100. Two images were obtained in the molecular layer of the dentate gyrus with two slices from each mouse analyzed. Puncta from the dentate gyrus were analyzed and counted using ImageJ, excluding cell somata. Hippocampal calbindin D28 images were obtained using a Zeiss Axio Imager Z1 fluorescence microscope with a 20× lens objective. Mosaic images of the entire hippocampus of each animal were obtained and analyzed. All histologic analyses were done using ImageJ and analyzed statistically by Student’s t-test, or by analysis of variance (ANOVA) with post hoc comparisons as indicated, using SPSS software (IBM, Armonk, NY, USA).

Larvae with melanotic masses (Fig. 1A) and melanized crystal cells (Figs 2G, 3H,J and 4H, Fig. S2F) were imaged with a Nikon SMZ1000 stereomicroscope. Still frames from movies of live larvae with GFP-labeled hemocytes (Fig. 2A) were taken with a Zeiss Axio Observer.Z1. Fixed hemocytes (Fig. 2E) were imaged with a Zeiss Axio Imager.Z1. Z-stacks of lymph glands were taken with a Zeiss Axio Imager.Z1 and analyzed using Zen software. Regions of interest (ROIs) surrounding the primary lymph gland lobes and 21 Z-positions (9.8 μm) surrounding the center Z-position were selected. With the exception of lymph glands marked with Trol (Fig. 2B, Fig. S3), constrained iterative deconvolution was applied to all lymph gland images prior to analysis of primary lobes (Figs 3B, 5A-F, 6B-G, 7A-D and 8E, Fig. S2A). All lymph gland images are presented as a single maximum-intensity projection.

The internalization of RNase A was visualized in B. pumilus. B. pumilus (5.5 log₁₀ cells/ml) in PBS were incubated with fluorescein isothiocyanate (FITC) labeled RNase A at 37 oC for 15 or 60 min as previously described [49 (link)]. Bacteria were washed three times with PBS to remove any unbound protein. After washing the bacteria is cultivated for 2 h in LB broth, washed to remove residual media components, and placed on a microscope slide for visualization. Fluorescence was monitored using a fluorescence microscope (Axio Imager Z1, Carl Zeiss, Germany). To visualize the internalization of RNase A, the biofilms of B. pumilus incubated with 100 µg/mL fluorescein-labeled RNase A were obtained as described earlier. After 24 h of growth at 37 °C, bacteria were washed three times with PBS to remove unbound proteins, and placed on a microscope to monitor the fluorescence using a fluorescence microscope (Axio Imager Z1, Carl Zeiss, Germany).

DIC microscopy was used to perform all the nucleolar imaging. Hypodermal, germ cell and pharyngeal muscle nucleoli of age-matched day 1 adults were imaged using 100X magnification with Axio Imager Z1 (Zeiss). Nucleolar area was quantified manually with the freehand tool using Fiji software. Details of the nucleolar size analysis are given in Supplementary Table 2. Worms carrying FIB-1::GFP and NCL-1::GFP transgenes were imaged using 63X magnification with Axio Imager Z1 (Zeiss). Immunofluorescent images were acquired using a laser-scanning confocal microscope (TCS SP5-X; Leica), equipped with a white light laser, a 405- diode UV laser, and a 100 × objective lens (HCX Plan-Apochromat CS 100 × oil, 1.46 NA). For human muscle biopsies, a total 15 representative fields with a 63X objective from each muscle sample were obtained, using the DM5500 fluorescent microscope (Leica) and the LAS AF software (version 2.3.6, Leica). Anti-Fibrillarin was detected with the Y5 cube, and nuclei were detected with the A4 cube. The area of the nucleolar and nuclear regions was quantified manually with the freehand tool, and subsequently the ratio of nucleolar/nuclear area was calculated. For the human samples, the average ratio of nucleolar/nuclear area (from an average of 100.4 (±28.9) nuclei) per sample was used for the analyses.

Cells were grown on glass coverslips or on polymer µ-slide IbiTreat (Ibidi). Thereafter, cells were fixed during 10 min with methanol, permeabilized and saturated with PBS-0.2%Triton-2% BSA and hybridized with various primary antibodies (p62, vimentin, desmin, actin, HspB5, MyHC) and Alexa Fluor secondary antibodies. Hoechst 33258 reagent was used to stain nuclei (5 min, 1 ng/mL). Observations were performed on Zeiss Axio Imager Z1 photomicroscope (Zeiss Inc., Oberkochen, Germany). Images were digitized with a camera (Coolsnap HQ2; Roper scientific) and acquired with Metavue Imaging system. Digitalization was done with Metavue software; images adjustments were performed on ImageJ.