Black and white images of oil Red O stained animals were captured using a 10X objective on a Zeiss Axio Imager microscope. Images were quantified using ImageJ software (NIH) as previously described (Noble et al., 2013 (link)). All reported results were consistent across biological replicates. Fluorescent images of reporters for FLP-7 secretion were captured using a 20X objective on a Zeiss Axio Imager microscope. The first pair of coelomocytes was imaged. mCherry fluorescence intensity in one of the two imaged coelomocytes was quantified and normalized to the area of the coelomocyte GFP as previously described and validated (Palamiuc et al., 2017 (link)). For fluorescence imaging of gene expression reporter lines (animals with integrated Patgl-1::GFP, Phsp-60::GFP, or Phlh-11::hlh-11GFP transgenes), an equal number of animals were chosen blindly and lined up side by side. Images were take using a 10X or 20X objective on a Nikon Eclipse 90i microscope. Fluorescence intensity for all chosen animals was quantified for each condition and normalized to area of the animals excluding the head as indicated in the figure legend. Images were quantified using ImageJ software (NIH).
Axioimager microscope
Manufactured by Zeiss
Sourced in Germany, United States, France, Australia, Canada, Japan, United Kingdom
The AxioImager is a high-performance microscope from Zeiss. It is designed for advanced imaging applications and provides reliable performance for a variety of research and clinical tasks. The AxioImager offers a range of optical systems and advanced imaging capabilities to meet the needs of users.
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Lab products found in correlation
Axiovision software, by Zeiss (20 mentions)
Dual spectral imaging fish system, by Applied Spectral Imaging (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (8 mentions)
Apotome, by Zeiss (8 mentions)
Gentra puregene kit, by Qiagen (7 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (7 mentions)
Propidium iodide, by Thermo Fisher Scientific (7 mentions)
Axiocam hrm camera, by Zeiss (6 mentions)
Lsm800 confocal microscope, by Zeiss (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (6 mentions)
Fluoromount g, by Southern Biotech (5 mentions)
Axiocam mrc5 camera, by Zeiss (5 mentions)
Axiocam mrm, by Zeiss (5 mentions)
Axiocam hrc camera, by Zeiss (5 mentions)
Matrigel, by BD (5 mentions)
Calcein am, by Thermo Fisher Scientific (4 mentions)
Chromosome analysis suite, by Thermo Fisher Scientific (4 mentions)
Fluoroshield mounting medium, by Abcam (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (4 mentions)
407 protocols using axioimager microscope
1
Quantitative Microscopy Imaging Protocols
2
Histological Analysis of Cellular Infiltration
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To analyze the cellular infiltration, 5-μM-thick sections were cut from paraffin-embedded tissue blocks and placed on charged microscope slides for staining. Sections were deparaffinized, rehydrated, and subjected to routine hematoxylin (Sigma) and eosin (Sigma) staining. Representative images were acquired using a Zeiss AxioImager microscope. To determine the phenotype of the cells at the injection site, 5-μM sections from paraffin-embedded tissues were cut, prepared, and blocked as described above. Endogenous peroxidase/phosphatase activity was blocked using Bloxall reagent (Vector Labs). Anti-F4/80 (6640; Abcam) and anti-CD3 (RM-9107; Thermo Scientific) as primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were used. Peroxidase activity was visualized using NovaRed HRP substrate (Vector Labs). Slides were then counterstained with hematoxylin, dehydrated with ethanol, and cleared with xylene substitute (Sigma). Coverslips were mounted with Organo/Limonene mounting medium (Sigma). Representative images were acquired using a Zeiss AxioImager microscope.
3
Quantifying Intestinal Lipid Droplets and Secretion
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Black and white images of oil Red O stained animals and fluorescent images were captured using a × 10 objective on a Zeiss Axio Imager microscope. In all cases, lipid droplet staining in the first four pairs of intestinal cells was quantified using NIH Image J software, by measuring pixel intensity over the first four pairs of intestinal cells, which fully encapsulate the variation seen in the tested conditions1 (link). For all atgl-1::GFP images, fluorescence intensity in the second and third pairs of intestinal cells was quantified. Fluorescent images of the reporters for FLP-7 secretion were captured using a × 20 objective on a Zeiss Axio Imager microscope. For all ‘secretion line' animals, the first pair of coelomocytes was imaged. mCherry fluorescence intensity in one of the two imaged coelomocytes was quantified and normalized to the surface area of the coelomocyte. Within each experiment, approximately 15–20 animals from each condition were quantified. All images were quantified using ImageJ (NIH).
4
Mapping Monosynaptic Circuit Inputs
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Nikon A1R Inverted or Nikon Upright Ni-E confocal optics were used to acquire images using a 20×/NA 0.75 Plan Apo VC or a 60×/1.4 NA objective. Data on starter cells were collected by analyzing z-stack images of all coronal sections spanning the entire injection site, acquired with A1R Nikon confocal microscopes, using the 'multipoint' function in Fiji (Schindelin et al., 2012 (link)). Mono-synaptic inputs were calculated as percent of total for each brain or normalized per starter cell, using Excel 365 (Microsoft) and GraphPad Prism 8 software.
The location and distribution of transsynaptically labeled neurons across the brain was assessed using a Zeiss AxioImager microscope using the 4×/0.10 Acroplan and 10×/0.3 Ph1 EC-Plan-NeoFluar objectives. Regions where the GFP+ somas were present were identified by comparison with the Paxinos and Franklin Mouse Brain Atlas. The Zeiss AxioImager microscope was also used to acquire overviews of coronal sections showing the brain-wide distribution of inputs, using a Plan NeoFluar 2.5×/0.075 objective.
The location and distribution of transsynaptically labeled neurons across the brain was assessed using a Zeiss AxioImager microscope using the 4×/0.10 Acroplan and 10×/0.3 Ph1 EC-Plan-NeoFluar objectives. Regions where the GFP+ somas were present were identified by comparison with the Paxinos and Franklin Mouse Brain Atlas. The Zeiss AxioImager microscope was also used to acquire overviews of coronal sections showing the brain-wide distribution of inputs, using a Plan NeoFluar 2.5×/0.075 objective.
5
Automated Microscopy Imaging System for Cell Analysis
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We have described TIS and the accompanying data analysis in previous publications29 (link),30 . The system used was the TIS basic 4 (pi4 Robotics GmbH, Berlin, Germany). It consists of a climate-controlled chamber containing: a Zeiss AxioImager microscope with a Colibri.2 lighting system and a Plan-Apochromat 63X/1.0 Ph3 M27 water immersion objective; an SC4022M digital imaging system (Finger Lakes Instrumentation, LLC, Lima, NY); and a robot-controlled motorized pipette. The TIS system included software programs (written by Reyk Hillert, Magdeburg, Germany) that were used to generate data for subsequent analysis. These included: Image Registrator v.1.1 (for image alignment and background subtraction); Binary Center v.1.0.2 (for binarization of images); MoPPi v.1.1.3.8 (converts binarized .pgn files into a single .xml file); and MultiCompare v.0.9.0 (extracts CMP data from .xml files). A flow chart for TIS image analysis is shown in Supplementary Fig. S1 .
6
Quantifying Tumor Hypoxia via Hypoxyprobe
7
Utricle Explant Immunostaining Protocol
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For utricle staining, explants were fixed in 4% paraformaldehyde for one hour and washed with PBS containing 0.1% TritonX-100. Primary antibodies in this study used were anti-Moysin7a (1:500, rabbit; Proteus), anti-Sox2 (1:500, rabbit; Millipore), anti-RFP (1:500, Rabbit; Millipore), anti-GFP (1:500, chicken; Abcam) and anti-CD326 (1:200; rat; Thermo Fisher; Catalog number 17-5791-82). Secondary antibodies used were Alexa Fluor 488 and Alexa Fluor 594 (1:2000, Invitrogen). Cell nuclei were labeled by incubation in 0.5 ug/mL of DAPI in PBS for 20 min. Immunofluorescence images were captured on a Zeiss AxioImager microscope with Apotome structured illumination..
8
Quantitative Muscle Fiber Analysis
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All sections were imaged using a Zeiss Axio Imager microscope at ×20 magnification. Images were tiled using Zen software. Fiber CSA was quantified on whole muscle cross sections using Myovision analysis software (45 (link), 46 (link)). eMyHC+ muscle fibers were quantified on whole muscle cross sections by a trained and blinded technician. Central nucleation was quantified on whole muscle cross sections using MuscleJ analysis software (47 (link)). γH2AX+ and p21+ nuclei were quantified from representative regions of interest (ROIs) using Myovision analysis software. Fibrosis was quantified with ImageJ software (NIH) using representative regions of interest from Masson’s trichrome–stained sections, as described previously (46 (link)).
9
Metabolic Biomarker Analysis in NAFLD
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The serum levels of TC, TG, HDL-C, LDL-C, ALT, AST, IL-1β, IL-6, and TNF-α were analyzed using serum biochemistry kits following the manufacturer’s specification. The procedures for the H&E and ORO-staining experiments were carried out as described in our previous study [22 (link)], and the images were captured using a Zeiss Axio Imager microscope. In addition, the NAFLD activity score (NAS) was used to measure the severity of NAFLD following the criteria reported by Kleiner et al. [35 (link)]. Total lipid was extracted from liver samples using ethyl alcohol (g:v (mL) = 1:9), and the hepatic TG and TC contents were detected according to the protocol described in previous work [23 (link)].
10
In Situ Hybridization of AqUFO Genes
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Fragments of AqUFO1 (383 bp) and AqUFO2 (328 bp) from non-conserved regions of the open reading frame were PCR amplified using primers listed in Supplementary Table S1
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