The frozen tissues were mounted on the specimen stages in the same way as described for “in situ hybridization”. Sections with 12 µm thickness were mounted onto a poly-l-lysine coated slide. Sections were fixed in cold 4% paraformaldehyde for 10 min after air drying. Slides were then processed for IF staining. IF was performed as previously described2 (link),4 (link). Frozen sections (12 µm) from each genotype were processed onto the same slides and incubated with primary antibodies listed in Supplementary Table 2 . For signal detection, secondary antibodies listed in Supplementary Table 2 were used. Nuclear staining was performed using Hoechst 33342 (5 µg/mL, H1399, Thermo Scientific). Pictures were taken using the Nikon Eclipse 90i upright microscope and processed by Nikon Elements Viewer.
Elements viewer
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The Elements Viewer is a software application designed for the analysis and visualization of elemental distribution data. It provides a user-friendly interface for displaying and interpreting the results of elemental analysis techniques such as X-ray fluorescence (XRF) or energy-dispersive X-ray spectroscopy (EDS).
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4 protocols using elements viewer
1
Immunofluorescence Staining of Frozen Tissue Sections
2
Mouse Sperm Morphology and Viability Analysis
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For male sterility experiments, 2-month-old littermate male mice were euthanized and relavent reproductive tissues were collected. Sperm were obtained by incubating a lacerated cauda epididymis in a pre-warmed HEPES-0.1% BSA buffer consisting of 130mM NaCl, 4mM KCl, 14mM fructose, 10mM HEPES, 1.35mM CaCl2, 1 mM MgCl2 in a droplet covered by embryo tested neat mineral oil. After incubating the cauda epididymis at 36°C for 30 min to allow the sperm cells to swim out, they were stained for a morphological and quantitative viability analysis. To stain the above sperm cells for a morphological and quantitative viability analysis, eosin-nigrosin staining was performed by using two parts 1% eosin Y (Sigma-Aldrich) and two parts 10% nigrosin (Sigma-Aldrich) well-mixed with one part mouse sperm cells. The resulting mix was then smeared on slides, and a coverslip applied with Cytoseal mounting media. Photographs of these slides were taken using a Nikon C2 DS-Ri1 color camera and analyzed using NIS Elements Viewer.
3
Fluorescence Confocal Microscopy of Aspergillus Biofilms
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Fluorescence confocal microscopy was performed on an Andor W1 spinning disk confocal with a Nikon Eclipse Ti inverted microscope equipped with a CFI Plan Fluor 20XC MI objective (Nikon). A. fumigatus and A. niger biofilms were cultured for imaging at 105 spores per ml in MatTek dishes (MatTek, P35G-1.0-14-C) in 2 ml of liquid glucose minimal medium for 24 h at 37°C with 5% CO2 in the dark. For visualization at 405 nm, biofilms were stained with 25 μg/ml calcofluor white (Fluorescent Brightener 28; Sigma, no. F3543) 15 min prior to imaging. The CFI Plan Fluor 20XC MI objective was used with water to image the bottom ∼300 μm of the biofilm with Z-slices collected every 1.2 to 1.5 μm. 3D rendering and image processing were performed in a Nikon Elements Viewer (Nikon). Quantification of biofilm architecture and generation of the heat map figures were carried out as previously described using the BiofilmQ framework written in MatLab (71 (link)). The framework is freely available for download (www.drescherlab.org/data ). All biofilm images and single columns in heat maps are representative of a minimum of three independent biological replicates.
4
Microscopic Imaging of Fungal Hyphae
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Spores were cultured at 104 spores/ml in 0.2 ml of liquid glucose minimal medium for 10 h at 37°C with 5% CO2 in the dark on MatTek dishes (MatTek, P35G-1.0-14-C). At this point, hyphae of various sizes had formed. Hyphae were imaged unfixed on an Andor W1 spinning disk confocal with a Nikon Eclipse Ti inverted microscope equipped with a CFI Plan Fluor 100× Oil objective (Nikon). 3D rendering and image analysis were performed in Nikon Elements Viewer (Nikon). For FM4-64 staining, hyphae were incubated in 10 μM FM4-64 in phosphate-buffered saline (PBS) for 15 min on ice and then briefly incubated in 37°C for 5 min before being washed twice with PBS and imaged as described above. Detection of B-glucan using Dectin-1 binding protocols was performed as previously published (72 (link)). Briefly, 12-h hyphal cultures were blocked in fluorescence-activated cell sorting buffer containing fetal bovine serum for 30 min, washed twice with PBS, and then incubated in 150 μl of soluble Dectin-1 for 1 h at room temperature. Hyphae were stained with goat anti-human IgG–Alexa Fluor 594 in PBS for 1 h at room temperature.
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