DIA muscles and left gastrocnemius muscles were frozen (cooled isopentane in N2; AGA) or fixed in paraffin (UNIBA), sectioned at 6–10 μm, and stained with hematoxylin and eosin (H&E). Endpoints included the assessment of the following parameters: degeneration, centronucleated fibers (CNFs), normal fibers, and regeneration. Five random digital images were taken using a Axioplan II and Axiocam HRC Color Camera on an Image Capture microscope (Zeiss, Jena, Germany) and blinded analysis was performed on data from at least 6 animals per group with Image J (U.S. National Institutes of Health, Bethesda, MD, USA) software (28 (link), 31 (link), 32 (link), 40 (link), 41 (link)).
Axiocam hrc color camera
Manufactured by Zeiss
Sourced in Germany
The AxioCam HRC is a high-resolution color camera designed for microscopy applications. It features a 6.45 megapixel CMOS sensor and supports a maximum resolution of 3,288 x 2,470 pixels. The camera provides fast image acquisition and can capture up to 15 frames per second at full resolution.
Automatically generated - may contain errors
Lab products found in correlation
Axioplan 2 microscope, by Zeiss (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Tissuefaxs plus system, by Zeiss (2 mentions)
Axiovision 4.1 microscope, by Zeiss (2 mentions)
Axio imager z2 microscope, by Zeiss (2 mentions)
Tissuefax, by TissueGnostics (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Axioplan 2, by Zeiss (1 mentions)
Stereo lumar v12, by Zeiss (1 mentions)
Shandon citadel 2000, by Thermo Fisher Scientific (1 mentions)
Hm 325, by Leica (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
X cite 120 mercury lamp, by Excelitas (1 mentions)
31000v2 dapi filter set, by Chroma Technology (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Axioscope microscope, by Zeiss (1 mentions)
H 3501, by Vector Laboratories (1 mentions)
Axioplan 2 bright field microscope, by Zeiss (1 mentions)
Ht110232, by Merck Group (1 mentions)
Pannoramic scanner, by 3DHISTECH (1 mentions)
12 protocols using axiocam hrc color camera
1
Histological Assessment of Muscle Degeneration
2
Zebrafish Transgenesis for Genetic Studies
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Microinjections of all four constructs were performed into one- to two-cell stage zebrafish embryos using 25–45 pg of plasmid DNA mixed with 50 pg of Tol2 mRNA. Embryos were incubated at 28° with 0.003% PTU (1-phenyl 2-thiourea) to suppress pigmentation. Embryos with transgene integration were identified by red fluorescent protein (RFP) expression in the skeletal muscle at 48 hr postfertilization (hpf), and green fluorescent protein (GFP) expression was observed in RFP positive embryos from 48 hpf to 5 d post fertilization (dpf). Embryos positive for both RFP and GFP were grown to adulthood. Several germline transmitting founders were identified for each construct, and their progeny were evaluated for patterns of GFP expression from 48 hpf to 5 dpf. Embryos from F2 generation of germline transmitting founders were also evaluated for GFP expression by crossing F1 adult fish with wildtype fish. The only difference in sequence between the C and T allele constructs was at the rs117273909 locus. Screening and imaging of embryos were performed using the Zeiss SteREO Lumar.V12 stereomicroscope with an AxioCam HRC color camera or Zeiss Axio Observer Z1 inverted microscope with an AxioCam MRm black and white camera.
3
Decalcification and Paraffin Embedding of Bone Tissues
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Tibias, femurs, and humeri were obtained and fixed for 48 h in Carson’s Millonig formalin at room temperature, and mineralized bone decalcification was made through EDTA protocol (Carson et al., 1973 (link)). Samples were processed in a Shandon Citadel 2000 tissue processor (Thermo, Waltham, MA, USA) according to standard histological techniques for paraffin embedding. Paraffin sections with a thickness of 5 μm were obtained in a rotary microtome (Microm HM-325 or Leica RT2125). These sections were de-waxed in xylol and hydrated in decreasing ethanol concentrations until being washed in distilled water to prepare for staining with H&E and Sirius red. Slides with sections from tibias, femurs, and humeri were analyzed in an Axiovert 200M microscope and the images were acquired with an AxioCam HRc color camera (Carl Zeiss, Oberkochen, Germany).
4
High-Resolution Cell Microscopy Imaging
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Each dispersed cell
population was
imaged using an AxioVert 200 fluorescence microscope (Carl Zeiss,
Oberkochen, Germany) with an X-CITE 120 mercury lamp (Lumen Dynamics,
Mississauga, Canada) and a 31000v2 DAPI filter set (Chroma Technology,
Irvine, CA). A 10× objective was used to obtain a mosaic image
with 10% overlap between neighboring images. Images were taken using
an AxioCam HRC color camera (Carl Zeiss) set to a resolution of 4164
× 3120.
population was
imaged using an AxioVert 200 fluorescence microscope (Carl Zeiss,
Oberkochen, Germany) with an X-CITE 120 mercury lamp (Lumen Dynamics,
Mississauga, Canada) and a 31000v2 DAPI filter set (Chroma Technology,
Irvine, CA). A 10× objective was used to obtain a mosaic image
with 10% overlap between neighboring images. Images were taken using
an AxioCam HRC color camera (Carl Zeiss) set to a resolution of 4164
× 3120.
5
Quantifying Tumor Markers with IHC
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The immunostaining assessment was performed by measuring the fraction by percentage (%) of means the HIF-1α, p53, BNIP3, Bcl-2, IAP-2, GLUT1, and Bax staining area in AMB and DF. Images collected from five random fields containing parenchyma and stroma obtained from each sample and acquired using an AxioScope microscope (Carl Zeiss, Oberkochen, DEU) equipped with an AxioCam HRC color camera (Carl Zeiss®). The images were acquired with a magnification of 400x. Diaminobenzidine-stained areas were separated and segmented. Then, they were analysed using immunohistochemistry (IHC) Image Analysis Toolbox of ImageJ Public domain software, developed by Wayne Rasband (NIMH, NIH, Bethesda, MD, EUA, http://rsbweb.nih.gov/ij/ ) [14 (link), 15 (link)]. After image segmentation, the percentage of tumour parenchyma-stained area was measured. Qualitative description of the location of the immunostaining in the plasma membrane, cytoplasm, and nucleus was carried out.
6
Paraffin Tissue Histological Staining
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Paraffin sections (10μ) were dewaxed in Histo-Clear (2 × 10 minutes), then rehydrated through a decreasing ethanol gradient (2 × 100%, 1 × 90/70/50/30% in ddH2O, 2 minutes each) and washed in dH2O for 2 × 5 minutes. For H&E staining, slides were incubated in hematoxylin (Sigma, MHSS32) for 3 minutes and then rinsed in at least 3 changes of tap water for a total of ten minutes. Next, slides were incubated in eosin (Sigma, HT110232) for 90 s, washed in ddH2O for 2 minutes, and allowed to dry overnight. The next day, coverslips were mounted using DPX mounting media (Millipore, 100579).
Alcian blue staining was performed using an alcian blue staining kit (Vector, H-3501). Briefly, sections were incubated in alcian blue solution (pH2.5) for 30 minutes and counter stained with nuclear fast red, as per the manufacturer’s instructions. Sections were imaged using a Zeiss Axioplan2 Brightfield microscope with Zeiss Axiocam HRc color camera.
Alcian blue staining was performed using an alcian blue staining kit (Vector, H-3501). Briefly, sections were incubated in alcian blue solution (pH2.5) for 30 minutes and counter stained with nuclear fast red, as per the manufacturer’s instructions. Sections were imaged using a Zeiss Axioplan2 Brightfield microscope with Zeiss Axiocam HRc color camera.
7
Immune Cell Quantification in Tissue
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Immunohistochemically stained slides were reviewed by two experienced pathologists to assess the morphology of inflammation and the quality of staining before digital image analysis.
After staining, whole slides were scanned using a high–performance Scanner (Pannoramic scanner, 3DHistech, Budapest, Hungary), and the scans were directly uploaded into the software (Qupath 0.3.2, open source software) used for digital image analysis based upon a machine learning approach. In brief, whole slides were annotated and a single threshold classifier was trained using a supervised machine learning classifier (random trees). As a training set five slides stained with CD3 and five slides stained with CD68 were selected to develop a classifier able to detect all immune cells.
Digital images of immunostained slices were acquired in AxioVision microscope software linked to an AxioCamHRc color camera and an AxioPlan 2 microscope (Zeiss, Jena, Germany). The immunohistochemical staining pattern was examined particularly for the mesenchymal surroundings.
After staining, whole slides were scanned using a high–performance Scanner (Pannoramic scanner, 3DHistech, Budapest, Hungary), and the scans were directly uploaded into the software (Qupath 0.3.2, open source software) used for digital image analysis based upon a machine learning approach. In brief, whole slides were annotated and a single threshold classifier was trained using a supervised machine learning classifier (random trees). As a training set five slides stained with CD3 and five slides stained with CD68 were selected to develop a classifier able to detect all immune cells.
Digital images of immunostained slices were acquired in AxioVision microscope software linked to an AxioCamHRc color camera and an AxioPlan 2 microscope (Zeiss, Jena, Germany). The immunohistochemical staining pattern was examined particularly for the mesenchymal surroundings.
8
Immunofluorescence Microscopy of Transfected Vero Cells
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At 24 hpt, transfected Vero cells cultured on glass coverslips (Thermo Scientific, Hampton, NH) were fixed in 3.7% formaldehyde for 20 min at room temperature, permeabilized for 20 min at room temperature with 0.1% Triton X-100, blocked for 30 min in blocking buffer, and stained with rabbit anti-p14 antiserum (1:200) or rabbit anti-myc antibodies (1:1000) or with 1:1000 dilutions of mouse monoclonal antibodies against a Golgi marker (PI4KIIIβ), TGN marker (TGN46), or ER marker (PDI) and with 1:1000 dilutions of Alexa 488–conjugated goat anti-mouse or Alexa 647–conjugated goat anti-rabbit secondary antibodies. Coverslips were mounted on glass slides using fluorescence mounting medium (Dako, Glostrup, Denmark) or Prolong gold antifade reagent (Life Technologies) and then visualized and photographed using a Zeiss LSM 510 META confocal microscope or a Zeiss Axioplan II MOT and AxioCam HRC Color Camera. Images were acquired with either 63 or 100× objective. Images were analyzed by ImageJ to generate fluorescence intensity graphs. Pearson's r was determined for 5–10 cells on unadjusted images using the Fiji version of ImageJ (Schindelin et al., 2012 (link)). If required, background was corrected using the rolling ball background plug-in, and colocalization thresholds were set using the Coloc_2 plug-in (Costes et al., 2004 (link)).
9
Histological Analysis of Myocardial Tissue
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Formalin-fixed tissues were paraffin-embedded and serially sectioned at 5 mm on a microtome. Basic myocardial histology and cellular infiltration were examined using heart cross sections stained with hematoxylin and eosin.
Immunohistochemistry for a-smooth muscle actin (SMA) (Sigma-Aldrich) was performed on sectioned paraffinembedded tissue that was deparaffinized and heat-treated for antigen retrieval before staining. In brief, endogenous peroxidases were quenched with 3% hydrogen peroxide, endogenous biotin was blocked (Dako biotin blocking system; Dako, Carpinteria, CA), and nonspecific staining was blocked with normal goat serum. Sections were incubated with primary antibody overnight at 4 C, followed by host-specific biotinconjugated secondary antibody. Antibody complexes were then conjugated to avidinebiotin complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) and developed using 3,3 0 -diaminobenzidine as the chromogen (Dako). Images were captured with an AxioCam HRC color camera (Carl Zeiss Microscopy, Jena, Germany).
Immunohistochemistry for a-smooth muscle actin (SMA) (Sigma-Aldrich) was performed on sectioned paraffinembedded tissue that was deparaffinized and heat-treated for antigen retrieval before staining. In brief, endogenous peroxidases were quenched with 3% hydrogen peroxide, endogenous biotin was blocked (Dako biotin blocking system; Dako, Carpinteria, CA), and nonspecific staining was blocked with normal goat serum. Sections were incubated with primary antibody overnight at 4 C, followed by host-specific biotinconjugated secondary antibody. Antibody complexes were then conjugated to avidinebiotin complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) and developed using 3,3 0 -diaminobenzidine as the chromogen (Dako). Images were captured with an AxioCam HRC color camera (Carl Zeiss Microscopy, Jena, Germany).
10
Quantitative Analysis of BDNF mRNA in Fetal Inner Ears
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All sections were digitally examined using Zeiss AxioVision 4.1 microscope software coupled to an AxioCam HRc color camera and an AxioPlan2 microscope (Zeiss, Jena, Germany).
The in situ probe localized sections were analyzed at ×20 magnification utilizing a TissueFaxs Plus System coupled onto a Zeiss ® Axio Imager Z2 Microscope. Analyzed sections were then acquired using the TissueFaxs (TissueGnostics ® , Vienna, Austria). The intensity of the BDNF probes localized on the sections was then evaluated using HistoQuest ® (TissueGnostics) software. Utilization of this software allows for an objective evaluation of the localized probes and is advantageous over a subjective assessment by the investigator. Nine fetal inner ears of different gestational ages were utilized for the BDNF mRNA quantification study. A list of the specimens and sections used is included in Table 3.
The in situ probe localized sections were analyzed at ×20 magnification utilizing a TissueFaxs Plus System coupled onto a Zeiss ® Axio Imager Z2 Microscope. Analyzed sections were then acquired using the TissueFaxs (TissueGnostics ® , Vienna, Austria). The intensity of the BDNF probes localized on the sections was then evaluated using HistoQuest ® (TissueGnostics) software. Utilization of this software allows for an objective evaluation of the localized probes and is advantageous over a subjective assessment by the investigator. Nine fetal inner ears of different gestational ages were utilized for the BDNF mRNA quantification study. A list of the specimens and sections used is included in Table 3.
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