In addition to histomorphological evaluation, serial sections were stained for assessment of collagen II and MMP‐13 contents. After deparaffinization and rehydration of the tissue sections, endogenous peroxidase activity was blocked by 3% H2O2 for 20 min. The proteins were immunostained using a 2‐step method according to the kit manufacturer's instructions. The sections were incubated with rabbit polyclonal anti‐collagen II antibody (ab34712, 1:100; Abcam, Cambridge, MA), and rabbit polyclonal anti‐MMP‐13 antibody (ab39012, 1:50; Abcam) overnight at 4°C. The slides were washed three times in PBS followed by incubation for 20 min at 37°C with an anti‐mouse/rabbit immunoglobulin G (IgG) detection system (PV‐9000; Zhongshan Goldenbridge Biotechnology Co., China) and visualized with diaminobenzidine. Nuclei were counterstained with hematoxylin for 5 min. The optical densities of the stained slides were measured using image analysis software (Nikon H600L Microscope and image analysis system, Japan). Collagen II was expressed by relative intensity. MMP‐13 was expressed by the percentage of positive cells.
H600l microscope and image analysis system
Manufactured by Nikon
Sourced in Japan
The Nikon H600L Microscope and Image Analysis System is a high-performance laboratory equipment designed for advanced microscopy and image processing applications. The system combines a robust and precise microscope with a powerful image analysis software, providing users with a comprehensive solution for a wide range of research and analytical tasks. The core function of the H600L is to enable detailed observation, examination, and digital capture of samples at high magnification levels, as well as to facilitate comprehensive image analysis and data processing.
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3 protocols using h600l microscope and image analysis system
1
Collagen II and MMP-13 Immunohistochemistry
2
Histological Evaluation of Hepatopancreas
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The xed hepatopancreas were dehydrated in an ethanol series (75% ~ 95%) and embedded in para n. Then, 5 µm-thick sections were cut and stained with hematoxylin-eosin (HE). Finally, observation and assessment of hepatopancreas were done on a light microscopy (Nikon H600L Microscope and image analysis system, Tokyo, Japan). Histological damages in the histopathological slices were quantitatively determined following the previous methods (Bernetet et al. 1999; Corbett et al. 2015) . Severity score value from 0 to 6 was assigned for the degree and extent of each alteration: 0-unchanged, (1 or 2)-mild, (3 or 4)moderate, (5 or 6)-severe.
3
Quantifying Cartilage Collagen and MMP-13
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After tissue section depara nization and rehydration, serial sections were stained for collagen II (COLII) and matrix metalloproteinase (MMP)-13 using a two-step method following manufacturer instructions. Sections were incubated overnight at 4°C with rabbit polyclonal anti-COLII antibody (ab34712, 1:100; Abcam) and rabbit polyclonal anti-MMP-13 antibody (ab219620, 1:50; Abcam). The slides were then washed three times in PBS, incubated for 20 min at 37°C with an anti-mouse/rabbit IgG detection system (PV-9000, Zhongshan Goldenbridge Biotechnology Co., China), and stained with diaminobenzidine (DAB). Nuclei were counterstained with hematoxylin for 5 min. The optical density of stained slides was measured using image analysis software (Nikon H600L Microscope and Image Analysis System, Japan). COLII was quanti ed by relative intensity. MMP-13 was quanti ed by the percentage of positive cells.
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