Fixed brains were cut into 40 μm sections using a Vibratome (Microm, France). Sections were permeabilized with 0.1% Triton X-100 in PBS, 2% Bovine Serum Albumin (BSA) and then incubated for 48h at 4°C with primary antibody (anti-IBA1 1:2000, Wako; anti-GFAP 1:500, Dako). After three washes in PBS, sections were incubated with the appropriate secondary antibody. Wide field or Apotome images of the whole unilateral hippocampus were acquired using an Imager Z1 microscope (Zeiss) equipped with an AxioCam MR3 camera. For analysis of IBA1 staining, for each field, 11 images corresponding to 10 μm-thick optical sections were acquired. For analysis of GFAP staining, for each field, 5 images corresponding to 4 μm-thick optical sections were acquired. Stacked images were merged and analyzed through the use of a custom developed ImageJ macro to determine (1) the number of immunopositive cells and mean cell body size (IBA1 staining) and (2) the percentage of immunopositive cell surface (GFAP staining). Fluoro-jade staining was performed according to manufacturer recommendations. Slices were examined with Imager Z1 microscope (Zeiss). The presence of positively stained neurons was observed in 6 sections randomly selected from the rostral and caudal portions of the hippocampus.
Imager z1 microscope
Manufactured by Zeiss
Sourced in Germany, United States, Canada
The Imager Z1 is a high-performance microscope designed for advanced imaging applications. It features a modular design, allowing for customization to meet specific research needs. The Imager Z1 provides high-resolution and high-contrast imaging capabilities, catering to a variety of scientific disciplines.
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Lab products found in correlation
Axiocam mrm, by Zeiss (7 mentions)
Fibronectin, by Merck Group (5 mentions)
Axiocam hrc camera, by Zeiss (4 mentions)
Axiovision 4, by Zeiss (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Zen software, by Zeiss (4 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Axiocam mrm camera, by Zeiss (3 mentions)
Fm4 64, by Thermo Fisher Scientific (3 mentions)
Axiocam hrm camera, by Zeiss (2 mentions)
Cisplatin, by Merck Group (2 mentions)
Axiocam mrm ccd camera, by Zeiss (2 mentions)
Mrm camera, by Zeiss (2 mentions)
Propidium iodide double staining, by Merck Group (2 mentions)
Annexin 5, by Merck Group (2 mentions)
Apo one homogeneous caspase 3 7 assay kit, by Promega (2 mentions)
Rm2255 microtome, by Leica (2 mentions)
Technovit 7100, by Kulzer (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Axiocam hrc, by Zeiss (2 mentions)
122 protocols using imager z1 microscope
1
Hippocampus Immunohistochemistry and Fluoro-jade Staining
2
Histological Assessment of Cardiac Fibrosis and Hypertrophy
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After euthanizing the mice, their hearts were extracted and subjected to a semi-quantitative assessment of fibrosis and cellular hypertrophy, following [12 (link)]. The samples were stained with Masson’s Trichrome or hematoxylin and eosin, and microphotographs were captured using an Imager Z.1 Zeiss microscope equipped with an AxioCam HRm camera. The microphotographs were processed using the AxioVision software. The PV records of mice with hearts that showed no signs of fibrosis and cellular hypertrophy indicative of heart failure were excluded from the study.
3
Quantitative FMDV Immunohistochemistry Protocol
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Immunohistochemistry was performed using an automated Discovery XT (Ventana Medical Systems, Roche Diagnostics), with streptavidin‐biotin‐peroxidase, 3′‐diaminobenzidine as a substrate and haematoxylin counterstaining. The primary antibodies were rabbit polyclonal against FMDV (FMDV O1 Manisa R3 262/97, The Pirbright Institute, United Kingdom) used at 1/1000; mouse monoclonal against FMDV non‐structural protein 3D polymerase (mAb 3F12, kindly provided by Dr E. Brocchi from IZSLER, Brescia, Italy) and mouse monoclonal against cytokeratin AE1‐AE3 (M3515, DAKO), used at 1/500.
Positivity for FMDV was evaluated by image analysis of sections stained with polyclonal antibodies. Scanned images of two sections per insert were taken by an ImagerZ1 Zeiss microscope and an AxioCam HRc Zeiss camera. FMDV‐positive cells and total number of cells (mean 600, SD 306 cells per insert) were manually counted on each section with ImageJ Cell Counter (Schneider, Rasband, & Eliceiri,2012 ).
Positivity for FMDV was evaluated by image analysis of sections stained with polyclonal antibodies. Scanned images of two sections per insert were taken by an ImagerZ1 Zeiss microscope and an AxioCam HRc Zeiss camera. FMDV‐positive cells and total number of cells (mean 600, SD 306 cells per insert) were manually counted on each section with ImageJ Cell Counter (Schneider, Rasband, & Eliceiri,
4
Cardiac Fibrosis and Hypertrophy Assessment
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The tissue remained in PFA 4% (wt/vol) in phosphate-buffered saline (PBS) for 24 hours at room temperature. The tissue was processed into a paraffin-embedded block and 3-μm slices were stained. Micrografts were acquired using a bright field microscope (Imager Z.1 Zeiss microscope, with AxioCa HRm). Images were analyzed with AxioVision software.
Fibrosis was assessed using Masson’s Trichrome staining, and images were acquired of whole tissue at 1.25 times and 5 times original magnification. Heart hypertrophy was assessed by normalizing heart weight to tibia length, and by analyzing hematoxylin and eosin (HE) staining in sliced tissues. Only cells with a nucleus at the center were considered. At least 20 cells per image were selected. The diameter was measured using Image J software (v1.53t, NIH).
Fibrosis was assessed using Masson’s Trichrome staining, and images were acquired of whole tissue at 1.25 times and 5 times original magnification. Heart hypertrophy was assessed by normalizing heart weight to tibia length, and by analyzing hematoxylin and eosin (HE) staining in sliced tissues. Only cells with a nucleus at the center were considered. At least 20 cells per image were selected. The diameter was measured using Image J software (v1.53t, NIH).
5
Quantifying Muscle Fiber and Stem Cell Dynamics
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Ten to fifteen images were recorded from each section with an Imager Z1 Zeiss microscope at 20x magnification connected to a CoolSNAP MYO camera for the quantification of the number of myonuclei per fiber and Pax7 + cells. Nuclei with their geometric center within the inner rim of the laminin ring were defined as myonuclei. The number of myonuclei was divided by the number of fibers analyzed on the same picture (Egner et al, 2016) . The number of Pax7 + cells was also divided by the number of fibers analyzed on the same picture (Theret et al, 2017) . For whole cryosection analysis, slides were automatically scanned at × 10 of magnification using an Axio Observer.Z1 (Zeiss) connected to a CoolSNAP HQ2 CCD Camera (photometrics). The image of the whole cryosection was automatically reconstituted in MetaMorph Software (Desgeorges et al, 2019) .
The number of IgG positive fibers (i.e., based on staining with cy3 anti-mouse), embryonic MyHC positive fibers and myofibers with central nuclei were quantified on the whole section and normalized to the total number of myofibers. Myofiber cross-sectional area (CSA) was determined on whole gastrocnemius muscle sections labeled by anti-laminin antibody using the Open-CSAM program, as previously described (Desgeorges et al, 2019) .
The number of IgG positive fibers (i.e., based on staining with cy3 anti-mouse), embryonic MyHC positive fibers and myofibers with central nuclei were quantified on the whole section and normalized to the total number of myofibers. Myofiber cross-sectional area (CSA) was determined on whole gastrocnemius muscle sections labeled by anti-laminin antibody using the Open-CSAM program, as previously described (Desgeorges et al, 2019) .
6
Immunostaining of Frozen Mouse Hearts
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Frozen OCT-embedded mouse hearts were sectioned on a cryostat with a section thickness of 8 µm. Air dried sections were fixed in ice cold acetone (−20 °C) for 10 min before a 1 h permeabilization step in PBS containing 0.1% saponin; 1% BSA; 4% donkey serum. Incubation with desired primary antibody was performed 1 h to overnight. Primary antibody signal was revealed using donkey anti-species fluorescent secondary antibody. Pictures were taken on Zeiss microscope imager Z.1 equipped with an apotome.
7
Immunostaining of Cryosectioned Mouse Hearts
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Frozen OCT-embedded mouse hearts were sectioned on a cryostat with a section size of 8 µm. Air dried sections were fixed in ice cold acetone (-20°C) for 10 min before a 1h permeabilization step in PBS containing 0.1% saponin; 1% BSA; 4% donkey serum. Incubation with desired primary antibody was performed 1h to overnight. Primary antibody signal was revealed using donkey anti-specie fluorescent antibody. Pictures were taken on Zeiss microscope imager Z.1 equipped with an apotome.
8
Imaging Live Larval Metamorphosis
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Live larvae were immobilized on slides coated with poly-L-lysine and imaged using a Zeiss Imager Z1 microscope using DIC optics. Larvae that were larger than the field of view at 5X (after 50 dpf) were photographed in two or three partial pictures that were later merged using the Adobe Photoshop CS6 Photomerge function. Metamorphosing larvae and juveniles were relaxed in a 1:1 mix of 7.5% MgCl2 and FSW prior to imaging.
9
Immunofluorescence Staining of Trypanosome Cells
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PCF detergent-extracted cells and permeabilized whole cells were prepared as described in [1 (link)] and fixed in 3% paraformaldehyde (PFA). The following primary antibodies were used diluted in PBS, supplemented with 0.1% Tween 20 (PBS-Tween 0.1%): (anti-BILBO1 rabbit polyclonal 1:6000), anti-TbKINX1B (home-made mouse IgM mAb specific to aa 820–1324, 1:4000), anti-FTZc (1:1000, [10 (link)]), anti-PFR2 (L8C4 1:10, [20 (link)]), and YL1/2 (Chemicon, MAB1864). Commercial secondary antibodies were secondary anti-mouse-FITC (Sigma F2012, 1:100), anti-rabbit Alexa 594 (Molecular Probes A11012, 1:100), anti-rat Alexa 488 or Alexa 594-conjugated for YL1/2 (Molecular Probes A21470 or 21471, dilution, 1:100). DNA was stained using DAPI. Images were acquired with Metamorph software on a Zeiss Imager Z1 microscope and processed by ImageJ. In the case of cells that were used for quantification of the phenotypes, BSF or PCF were fixed in methanol for 30 min and further used for immunofluorescence using PFR2 marker L8C4. Acquisition of images using 63× objective and random slide auto-focus application was employed using Metamorph software. A minimum of 200 cells in each case was counted, in three independent experiments (n = 3) and results were plotted using Prism Software, Version 5.
10
Immunofluorescence Imaging of IMCD-3 Cells
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IMCD-3 cells were fixed with 4% PFA, permeabilized with 0.15% Triton X-100 in PBS, and blocked with blocking buffer (1% BSA and 0.05% Tween-20 in 1x PBS) for 45 minutes at room temperature (RT). Cells were incubated with primary antibodies (in blocking buffer) for 1 hour at RT, and washed three times with PBS tween (0.05% Tween-20 in 1x PBS). Cells were incubated with fluorescent-conjugated secondary antibodies (in blocking buffer) for 45 minutes at RT, and washed three times with PBS tween. After the washing steps the cells were washed with 70% ethanol for 1 minute and 100% ethanol for 1 minute. The samples were air-dried and mounted on a microscope slide with mounting solution (20 mM Tris HCl pH 8, 0.2 M DABCO, 90% glycerol). Subcellular localization studies and cilia length measurements were performed using a Zeiss Imager Z1 microscope with a 63x (NA 1.4) objective.
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