Twelve fields, with an area of 400,000 µm2, were randomly selected from each section and were analyzed for the absorbance of each dye including van Gieson, Mallory, and Alcian blue staining. The sections of cartilage images were analyzed with image analysis software (AxioVision Release 4.8.2-SP2 Software, Carl Zeiss Microscopy GmbH, Jena, Germany), which quantifies the level of dye absorbance in the colorimetric count (pixel2)/μm2. Through a blinded review, two experienced histologists evaluated obtained photographs in accordance with previously published protocol33 (link). For histomorphometry, the computerized imaging analytical software (AxioVision Release 4.8.2-SP2 Software, Carl Zeiss Microscopy GmbH, Jena, Germany) was used as previously described33 (link).
Axiovision release 4 8 2 sp2
Manufactured by Zeiss
Sourced in Germany
AxioVision Release 4.8.2-SP2 Software is a comprehensive imaging and analysis software solution for microscopy applications. It provides tools for image capture, processing, analysis, and documentation.
Automatically generated - may contain errors
16 protocols using axiovision release 4 8 2 sp2
1
Quantitative Histomorphometric Analysis of Cartilage Dyes
2
Histochemical Quantification of Cartilage Repair
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One field of about 550,000 µm2, corresponding to the defect area, carefully selected from each section (three sections for each time point), was analysed for histochemical assessment of Alcian Blue staining, detecting GAGs expression, and to quantify the level of positive anti-Collagen I, anti-Collagen II, anti-Aggrecan and anti-SOX9 antibodies immunoexpression. The image analysis software (AxioVision Release 4.8.2-SP2 Software, Carl Zeiss Microscopy GmbH, Jena, Germany), which quantifies the staining level as the densitometric count (pixel2) normalized to the defect area of each sample, was used. The samples were analysed by using the Zeiss Axioplan light microscope (Carl Zeiss, Oberkochen, Germany) and the pictures were taken with a digital camera (AxioCam MRc5, Carl Zeiss, Oberkochen, Germany). Two investigators (one anatomical morphologist and one histologist) made the morphological assessment. If disputes occurred, a unanimous agreement was reached after section re-evaluation and before proceeding with data interpretation.
3
Quantifying Immunohistochemistry Staining Intensity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Image analysis software (AxioVision Release 4.8.2-SP2 Software, Carl Zeiss Microscopy GmbH, Jena, Germany), which quantifies the level of staining intensity of positive immunolabelling, was used to calculate the percentage area stained with CHI3L1 and lubricin antibodies in 15 fields, randomly selected from each section. Digital micrographs were taken using the Zeiss Axioplan light microscope (Carl Zeiss, using objective lens of magnification ×20 i.e., total magnification 400) fitted with a digital camera (AxioCam MRc5, Carl Zeiss). Three blinded investigators (2 anatomical morphologists and one histologist) made the evaluations that were assumed to be correct if values have not statistically significant difference [57 (link)]. If disputes concerning interpretation occurred, unanimous agreement was reached after sample re-evaluation.
4
Morphometric Analysis of Muscle Fibers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Seven fields, the total area of which was about 150,000 µm2, randomly selected from each muscle (proximal area of anterior tibial of leg of right hind limb) cross section, were analyzed for morphometric analysis. The perimeter of the muscle fibers was considered and calculated using a software for image acquisition (AxioVision Release 4.8.2-SP2 Software, Carl Zeiss Microscopy GmbH, Jena, Germany). Negative images were used for a better software performance in the morphometric analysis. The data were expressed as mean ± standard deviation (SD). The statistical significance of the results was then evaluated. A Zeiss Axioplan light microscope (Carl Zeiss, Oberkochen, Germany) fitted with a digital camera (AxioCam MRc5, Carl Zeiss, Oberkochen, Germany) was used to take digital micrographs; the evaluations were made by three blinded investigators, whose evaluations were assumed to be correct if the recorded values were not significantly different. In case of dispute concerning interpretation, the case was reconsidered to reach a unanimous agreement [18 (link)].
5
Quantitative MMP-7 and MMP-9 Immunohistochemistry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The MMP-7 and MMP-9 antibody-staining status were identified as either negative or positive. Immunohistochemical positive staining was defined by the presence of brown chromogen detection on the edge of the hematoxylin-stained cell nucleus, distributed within the cytoplasm or in the membrane via evaluation by light microscopy as previously described.28 (link) Positive controls consisted of tissue specimens with known antigenic positivity. Sections treated with PBS without the primary antibodies served as negative controls. Seven fields of about 600.000 μm2, randomly selected from each section, were considered for morphometric and densitometric analysis. The percentage areas (morphometric analysis) stained with MMP-7 and MMP-9 antibodies were expressed as % positive, dark brown pixels of the analyzed fields. While, the levels (high/low) of staining intensity of positive areas (densitometric analysis) were expressed as densitometric count (pixel2) of positive, dark brown pixels of the analyzed fields. These parameters were calculated using software for image acquisition (AxioVision Release 4.8.2 - SP2 Software, Carl Zeiss Microscopy GmbH, Jena, Germany). Data were expressed as mean ± standard deviation (SD). Digital micrographs were taken and fitted as previously described.
6
Quantifying Cellular Pellet Morphology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The size of the pellets was analyzed at 7, 14, 21, and 28 days. The cellular pellets presented an ellipsoidal shape and their surface at different time points was calculated as follows: the histological sections (objective lens: 2.5×) of the middle part of pellets, stained by hematoxylin and eosin (H&E) have been used. The pellet surface has been calculated by using an image-analysis software (AxioVision Release 4.8.2-SP2 Software, Carl Zeiss Microscopy GmbH, Jena, Germany) and expressed in μm2. Image analysis was performed on four pellets per condition.
7
Histochemical and Immunohistochemical Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four pellets per group, the area of each of which was about 3,500,000 Pixel2, were analyzed for histochemical evaluation of Alcian Blue staining, which detects mucosubstance content (GAGs) and to quantify the level of immunostaining of positive anti-Col I, anti-Col II, anti-Col X, anti-aggrecan, anti-SOX9, and anti-lubricin antibodies immunolabeling. It was used as an image-analysis software (AxioVision Release 4.8.2-SP2 Software, Carl Zeiss Microscopy GmbH, Jena, Germany), which quantifies the level of staining in the densitometric count (pixel2) normalized to the area of each section expressed in pixel2. Digital micrographs were taken using the Zeiss Axioplan light microscope (Carl Zeiss, Oberkochen, Germany), using a lens with a magnification of ×10, i.e., total magnification 100) fitted with a digital camera (AxioCam MRc5, Carl Zeiss, Oberkochen, Germany). Three blinded investigators (two anatomical morphologists and one histologist) made the evaluations that were assumed to be correct if the recorded values had no statistically significant difference. If disputes concerning interpretation occurred, a unanimous agreement was reached after sample re-evaluation.
8
Immunohistochemical Analysis of ADNP and β-Tubulin III
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression and distribution of ADNP and β-Tubulin III in the DG and cerebellum of sedentary and PA rats was evaluated through immunohistochemical analysis as previously described [53 (link)]. The sections were incubated overnight at 4 °C with the specific antibodies: rabbit anti-ADNP (NBP1-89236); mouse anti- β-Tubulin III (ab78078). The immunoreaction was visualized using a 3,3′-diaminobenzidine solution (DAB substrate Kit; SK-4100, Vector Laboratories, Burlingame, CA, USA). Following this, the samples underwent a light counterstaining with hematoxylin. Observation was carried out utilizing an Axioplan Zeiss light microscope (Carl Zeiss), and the digital micrographs were captured using a digital camera (AxioCam MRc5, Carl Zeiss) via AxioVision Release 4.8.2—SP2 Software (Carl Zeiss Microscopy GmbH, Jena, Germany). Original microphotographs are reported in Figure S1 (scale bar 50 μm and 10 μm).
The percentage of area stained with β-Tubulin III or ADNP antibody was calculated by using ImageJ software (NIH, Bethesda, MD; available athttp://rsb.info.nih.gov/ij/index.html , accessed on 9 January 2024), which quantifies the level of staining intensity of positive immunolabeling in each field. The statistical analysis details are reported in Table S1 .
The percentage of area stained with β-Tubulin III or ADNP antibody was calculated by using ImageJ software (NIH, Bethesda, MD; available at
9
Quantitative Analysis of Inflammatory Markers in Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Image analysis software (AxioVision Release 4.8.2-SP2 Software, Carl Zeiss Microscopy GmbH, Jena, Germany), which quantifies the level of staining of positive anti-IL-1β, anti-IL-4, anti-IL-6, anti-IL-10, anti-TNF-α, anti-MMP-13, and anti-lubricin antibodies immunolabelling, was used to calculate the densitometric count (pixel2) in seven fields, area of which was about 150.000 µm2, randomly selected from each section. Digital micrographs were taken using the Zeiss Axioplan light microscope (Carl Zeiss, using objective lens of magnification 20× i.e., total magnification 200×) fitted with a digital camera (AxioCam MRc5, Carl Zeiss). Three blinded investigators (two anatomical morphologists and one histologist) made the evaluations that were assumed to be correct if values have not statistically significant difference [54 (link)]. If disputes concerning interpretation occurred, unanimous agreement was reached after sample re-evaluation.
10
Muscle Fiber Morphometric Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each H&E stained muscle cross section was subjected in triplicate to morphometric analysis by calculating the area of twenty muscle fibers of five randomly selected fields with a total area of about 35.000 μm2, using a software for image acquisition (AxioVision Release 4.8.2—SP2 Software, Carl Zeiss Microscopy GmbH, Jena, Germany) [60 (link)]. Data were then expressed as diameter mean ± standard deviation (SD). Statistical significance of results was thus accomplished. Three investigators (two anatomical morphologists and one histologist) made the morphological assessment. If disputes occurred, a unanimous agreement was reached after section re-evaluation and before proceeding with data interpretation.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!