The mice were sacrificed after OGTT and hormone test. The pancreas was quickly removed and fixed in 4% paraformaldehyde for 24 h. After ethanol gradient dehydration and xylene immersion, the mice were embedded in paraffin, marked with clipping, and prepared for sections. Five-micron thick sagittal sections were used and randomly selected for Hematoxylin and Eosin (HE) staining. As previously described, a three-step avidin-biotin complex staining procedure was taken (Wang et al., 2014 (link)). The pancreas sample was performed using insulin polyclonal antibody (1:1,000, Abcam) and glucagon (1:1,000, Abcam) to detect the changes of insulin and glucagon expression in islets. The insulin and glucagon-positive cell area was stained orange and other negative cell area of islet was pale. HE-stained sections were analyzed under a light microscope (Leica DM2500, Wetzlar, Germany), and images were acquired using a Leica DFC 490 system (Leica Microsystems, Wetzlar, Germany). Using Photoshop software (Adobe Photoshop CS3, Adobe, CA, USA), the percentage of the insulin and glucagon-positive area relative to the whole area of the islet was calculated (n = 6), and the average value was used to represent the positive area percentage for each sample.
Dfc 490 system
Manufactured by Leica
Sourced in Germany
The Leica DFC 490 is a digital camera system designed for microscopy applications. It features a high-resolution sensor and advanced imaging capabilities to capture detailed micrographs. The camera is compatible with a variety of Leica microscopes and can be integrated into various imaging workflows.
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4 protocols using dfc 490 system
1
Pancreas Histological Analysis Protocol
2
Quantitative Evaluation of Temporomandibular Joint Cartilage and Subchondral Bone
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For conventional hematoxylin and eosin (H&E) staining, Leica DFC490 system (Leica Microsystems, Wetzlar, Germany) was used for collection of microscopic images. The condyle surface was divided into the front, middle and rear parts with articular disc front and rear joint as the boundary, quarter each part. The Leica QWin software (Leica Microsystems Imaging Solution, Ltd., Cambridge, UK) was applied to measure the fiber layer thickness, calcified cartilage layer thickness and full-layer cartilage thickness at a total of 9 positions in each quartering point and then taking the mean value for analysis. The Micro-CT (GE Healthcare, London, ON, Canada) was used to scan the specimen with the resolution of 8 µm and the peak voltage of 80 kV. Microview software (version 2.5.0; GE Healthcare) was used to re-construct the condylar specimen axial, coronal and sagittal images and to calculate the bone mineral density (BMD) and bone microstructure parameters [bone surface area-to-volume ratio (BS/BV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular bone gap (Tb.Sp)] of each specimen. The 0.5×0.5×0.5 mm region of interest (ROI) at the midpoint of the condyle postmedian was selected and just below the boundary of the cartilage and subchondral bone software was used to automatically calculate subchondral bone-related parameters in the region of interest.
3
Immunohistochemical Analysis of Craniofacial Tissues
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Haematoxylin and eosin (HE) staining was performed as we previously reported.15 Images were obtained with a Leica DFC490 system.
For IHC staining, a standard, three‐step, avidin‐biotin complex staining procedure was performed with anti‐EGR1 (1:100, 4153, Cell Signalling Technology, MA, USA), anti‐Exph1 (1:200, orb340819, Biorbyt LLC, CA, USA) and anti‐IL10 (1:200, ab192271, abcam, OR, USA) primary antibodies to detect changes in lower anterior alveolar bone and TMJs including disc, condylar cartilage and subchondral bone to detect the location of the three proteins.
For IHC staining, a standard, three‐step, avidin‐biotin complex staining procedure was performed with anti‐EGR1 (1:100, 4153, Cell Signalling Technology, MA, USA), anti‐Exph1 (1:200, orb340819, Biorbyt LLC, CA, USA) and anti‐IL10 (1:200, ab192271, abcam, OR, USA) primary antibodies to detect changes in lower anterior alveolar bone and TMJs including disc, condylar cartilage and subchondral bone to detect the location of the three proteins.
4
Quantifying Cx43-Positive Cells in TMJ Cartilage
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