Following autopsy, histopathological examination was performed on dorsal skin tissue using hematoxylin and eosin (H&E) and Masson’s trichrome stain. Stained tissues were photographed using AxioVision SE64 (ZEISS, Oberkochen, Germany) and analyzed using Carl Zeiss AxioVision SE64 Rel. 4 software to measure epidermal thickness.
Axiovision se64
Manufactured by Zeiss
Sourced in Germany
AxioVision SE64 is a software solution for microscope image acquisition, processing, and analysis. It provides a user-friendly interface for controlling various microscope components and capturing high-quality digital images. The software supports a range of microscope techniques and enables seamless integration with Zeiss microscope hardware.
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Lab products found in correlation
Axiocam mrc5, by Zeiss (9 mentions)
Axiophot photomicroscope, by Zeiss (6 mentions)
Axio observer z1 microscope, by Zeiss (4 mentions)
Axiocam mrm camera, by Zeiss (4 mentions)
Stereo discovery v20, by Zeiss (3 mentions)
Axioimager inverted z1, by Zeiss (3 mentions)
Axio imager, by Zeiss (2 mentions)
Environmental chamber, by Zeiss (2 mentions)
Axiovert 200m inverted microscope, by Zeiss (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Axioskop 2 plus, by Zeiss (2 mentions)
Spss statistics program for windows, by IBM (1 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Fitc phalloidin, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Axiocam mrm digital camera, by Zeiss (1 mentions)
M205a stereomicroscope, by Leica camera (1 mentions)
Ab51254, by Abcam (1 mentions)
Axio scope optical microscope, by Zeiss (1 mentions)
Laser scanning confocal microscope, by Nikon (1 mentions)
45 protocols using axiovision se64
1
Histopathological Analysis of Dorsal Skin
2
Quantitative Analysis of Cell Proliferation
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The anti-PCNA or anti-BrdU immunostaining slides were examined with AxioCam MRc5 (Carl Zeiss) interfaced with an Axiophot Photomicroscope (Carl Zeiss). Twenty randomly selected fields from each group were photographed and the AxioVision SE64 (Carl Zeiss) program was used for analysis. Within each image, the number of anti-PCNA or anti-BrdU positive cells or nuclei and the total number of muscle fibres was counted. The PCNA or BrdU ratio was analyzed as the ratio of the number of anti-PCNA/anti-BrdU positive cells or nuclei to the 1,000 muscle fibres.
3
Immobilization-induced Muscle Fiber Analysis
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A previous study reported that type 1 muscle fibre was predominantly affected in immobilization-induced GCM atrophy than type 2[13 (link)]. Therefore, type 1 muscle fibres were analysed in the current study. The sections were immunohistochemically stained for type 1 muscle fibres using monoclonal anti-myosin antibodies (skeletal, slow; Sigma-Aldrich). All histologic analyses were performed by an anatomist who was blinded to the group allocation throughout the study. At the time of study, the anatomist had 20 years’ experience of performing histologic examination. The sections were examined with the Axiophot Photomicroscope (Carl Zeiss) and the images of 5 randomly selected fields from each group were captured with the AxioCam MRc5 (Carl Zeiss). The entire muscle cross section was determined from digital images (×100) of the anti-myosin immunostained muscle sections. The cross sectional area (CSA) of the anti-myosin positive type 1 muscle fibre was traced using the image morphometry program (AxioVision SE64; Carl Zeiss), and the average CSA of the type 1 muscle fibre was measured.
4
Quantifying BrdU-Positive Cells in Augmented Sinus
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The number of BrdU-positive nuclei or cells was counted. Ten randomly selected fields from each group were photographed, and the AxioVision SE64 (Carl Zeiss) program was used for analysis. The BrdU-labeled cells were counted in soft tissues, including fibrous tissue, vascular tissue, and bone marrow of the augmented sinus, and calculated per 1 mm2 of soft tissue of the augmented sinus. Statistical analyses were performed with the IBM SPSS Statistics program for Windows (ver. 19.0; IBM, Armonk, NY, USA). In addition to standard descriptive statistical calculations (means and standard deviations), ANOVA was used to determine intra- and intergroup statistical differences. When ANOVA yielded significant results, indicating that the group was significantly different from the others, Tukey’s test was performed. Mean values are accompanied by 95% confidence intervals, and all data are expressed as mean±standard deviation. Statistical significance was noted at P<0.05.
5
Axio Observer Z1 GFP Imaging
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Images were acquired using an Axio Observer Z1 microscope and AxioVision SE64 (release 4.8) software (Carl Zeiss). All phenotypes shown in Figure 4 , GFP-positive (+) or GFP-negative (−), were fully penetrant and observed for ≥10 animals per condition.
6
Visualizing Bacterial Viability
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After cultured in MH broth as mentioned above, C. jejuni samples (100 μl) were taken at 0 h and after 12 h culture. Samples were washed twice with PBS and stained with the LIVE/DEAD BacLight Bacteria Viability Kit (Life Technologies). Samples were observed with a fluorescence microscope (Carl Zeiss M100, Germany), and image analysis was performed with AxioVision SE64 (Version 4, Carl Zeiss).
7
Immunofluorescence Analysis of Endothelial Cell Junctions
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HLMVEC (1 × 105 cells) were seeded onto Lab‐Tek II chamber slides (Nalge Nunc International) and grown until 90% confluency was obtained as previously described.25 Twenty‐four hours after treatment with cytomix with or without HMW or LMW HA, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. After additional washing, the cells were permeabilized by 0.1% Triton X‐100 and then blocked with 1% BSA at room temperature. For F‐actin staining, the slides were incubated with FITC‐phalloidin (Thermo Fischer Scientific) for 30 min at 37°C. For ZO‐1 staining, slides were incubated with Alexa ZO‐1 antibodies (5 μg/ml, Thermo Fischer Scientific) for 1 h at room temperature. For VE‐cadherin staining, primary antibodies to VE‐cadherin (1:100, Cell Signaling) were used. After multiple washing with PBS, slides were then incubated with the secondary antibody Alexa Fluor 488‐conjugated Goat Anti‐Rabbit IgG (H1L) DS Grade (1:50, Invitrogen, Thermo Fischer Scientific) for 1 h at room temperature. Slides were then mounted with Vectashield with DAPI mounting medium (Vector Laboratories). Images were obtained by AxioVision SE64 (Carl Zeiss Microscopy, LLC).
8
Giemsa Staining of Heme-Containing Cells
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Cells were from either peripheral blood, obtained through tail snip, or spleen or liver single cells isolated and passed through a 100-micron pore size nylon mesh. In some experiments, CD11bhi/CD11c− cells were sort purified from T cell and B cell depleted spleen samples. Blood samples were smeared on a glass slide and allowed to dry, spleen and liver cells were placed onto a glass slide in a drop of RPMI containing 15% mouse serum and incubated at 37°C with 5% CO2 for 30 min to allow cells to adhere. For all samples, cells were fixed in methanol for 20 min and stained with Giemsa dye (Azer Scientific, Morgantown, PA) for 20 min and rinsed with ddH2O. In some cases, cells were incubated with Hz as described in above and Hz is visible as brown pigment, indicated with arrows. Images were obtained with a Nikon eclipse Ci light microscope at 100X magnification or a Zeiss Axio Imager. A1 microscope with an AxioCam MRm digital camera (Zeiss) at 63 or 96X in oil emersion. Scale bars were estimated based on average diameter of murine erythrocytes (Nikon) or with Axiovision SE64 imaging software (Carl Zeiss Microscopy, LLC, White Plains, NY).
9
Quantifying Angiogenic Markers in Muscle Tissue
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We used an AxioCam MRc5 (Carl Zeiss) interfaced with an Axiophot Photomicroscope (Carl Zeiss) to examine anti-PCNA, VEGF, and PECAM-1 immunostaining slides. We randomly photographed twenty chosen areas from the groups and analyzed them with the AxioVision SE64 (Carl Zeiss) program [24 (link), 25 ]. On each image, we counted the total number of anti-PCNA-, VEGF-, and PECAM-1-positive cells or nuclei as well as the total number of muscle fibers. The PCNA, VEGF, and PECAM-1 ratio was then calculated by multiplying the number of anti-PCNA-, VEGF-, and PECAM-1-positive cells or nuclei by 1,000 muscle fibers.
10
Semiquantitative Immunohistochemical Analysis
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Thirty randomly selected fields from each group were photographed using AxioCam MRc5 interfaced with Axiophot Photomicroscope. AxioVision SE64 (Carl Zeiss) program was used for analysis. A semiquantitative scoring system was used for nuclear or cytoplasmic markers PCNA, VEGF, and PECAM-1, considering the staining intensity and extent of area. This approach is widely accepted, and has been used in previous studies19 (link),20 (link). Briefly, the proportion of positive stained cells was scored as 0 (no cells stained positive), 1 (1–10% stain-positive cells), 2 (11–33% stain-positive cells), 3 (34–66% stain-positive cells), or 4 (67–100% stain-positive cells). The intensity of COL-1 immunostaining or MT staining was classified as 0 = negative staining, 1 = slight positive staining, 2 = moderately positive staining, or 3 = strongly positive staining.
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