Scaring tissues from patients or mice are fixed, dehydrated, embedded, and cut according to conventional methods. Hematoxylin and eosin (H&E) and Masson's trichrome staining are conducted to observe histological change and ECM deposition. For immunohistochemistry staining, the slides are incubated with the primary antibodies against col I (1 : 100, Abcam, UK), col III (1 : 100, Abcam, UK), and α-SMA (1 : 200, Abcam, UK) overnight at 4°C. Next, the slides are incubated with biotinylated antibody (Histostain™ kit, ZSGB, China) for 10 min at room temperature and visualized with diaminobenzidine (ZSGB, China). The slides are examined by FSX100 Bio Imaging Navigator (Olympus, Japan).
Fsx100 bio imaging navigator
Manufactured by Olympus
Sourced in Japan
The FSX100 Bio Imaging Navigator is a microscope imaging system designed for biological research. It provides high-quality image capture and analysis capabilities for a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Pv6000 histostain kit, by ZSGB-BIO (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
α sma, by Abcam (1 mentions)
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Microm hm550, by Thermo Fisher Scientific (1 mentions)
Neg 50tm, by Thermo Fisher Scientific (1 mentions)
Image pro plus 6.0 system, by Media Cybernetics (1 mentions)
14 protocols using fsx100 bio imaging navigator
1
Histological Analysis of Scar Tissue
2
Phagocytosis of FAM-labeled Amyloid-β
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Phagocytosis was examined via FSX100 Bio Imaging Navigator (Olympus Waltham, MA) using a Human β-Amyloid (1–42), 5-FAM-labeled according to the manufacturer’s protocol. Primary microglial cells cultured in glass-based dishes were used. Human β-Amyloid (1–42), 5-FAM-labeled was reconstituted and diluted with 1X PBS and to a concentration of 3 µg/mL according to both the manufacturer’s protocol and previous reports40 (link),41 (link). We incubated the cells in standard culture conditions for 3 h. After aspirating the culture medium, the cells were fixed with 4% paraformaldehyde. Then, after discarding paraformaldehyde, we washed fixed cells with 1 mL phosphate buffered salts (PBS) twice and measured the fluorescence intensity of FAM along the long axis of the cytoplasm using an Imaging Navigator.
3
Intracellular ROS Detection with DCFH-DA
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A relatively specific probe for hydrogen peroxide, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), was used to analyze the formation of intracellular ROS. cells were incubated with 2.4 mM DCFH-DA (5 μL) for the final 30 min of the treatment. Then, cells were washed twice with PBS. For visualization of the intracellular fluorescence, the cells were observed with a FSX100 Bio Imaging Navigator, which is an all-in-one fluorescence imaging system (Olympus Corporation, Tokyo, Japan). The intracellular ROS production was evaluated via measurement of the fluorescence intensity.
4
Histological Evaluation of AAV5-Mediated Gene Delivery
5
Histological and Immunohistochemical Analysis
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The samples were fixed with 4% paraformaldehyde, dehydrated in graded ethanol, embedded in paraffin, and then cut into 5-μm-thick sections. H&E and Masson’s trichrome staining were used to detect the histological change and collagen deposition. For immunohistochemistry staining, the sections were immersed in 3% H2O2 after deparaffinization to eliminate the activity of endogenous peroxidase at 37 °C for 15 min and blocked with 5% BSA in PBS for 1 h to exclude the non-specific binding. Then, the slides were incubated with the primary antibodies against α-SMA and IL-17RA overnight at 4 °C. The next day, the slides were incubated with a PV6000 Histostain™ kit (ZSGB, Beijing, China) and stained with diaminobenzidine (ZSGB, Beijing, China). Images were obtained by FSX100 Bio Imaging Navigator (Olympus, Tokyo, Japan).
6
Intracellular ROS Quantification using DCFH-DA
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Intracellular ROS formation was analyzed using 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) (Tamura et al., 2013 ). Briefly, 2.4 mM DCFH‐DA (5 μl) was added to the medium before 30 min of the treatment. After washed with phosphate‐buffered saline (PBS) twice, HSCs were observed under a fluorescence imaging system (FSX100 Bio Imaging Navigator, Olympus Corporation). The fluorescence intensity of intracellular ROS was measured by ImageJ.
7
Histochemical detection of ROS
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Histochemical staining for the presence of hydrogen peroxide and superoxide was performed following treatments according to the procedures described in Thordal-Christensen et al. (1997 (link)) and Dunand et al. (2007 (link)), respectively, with slight modifications for each of them. To evaluate the presence of H2O2, roots were stained with 1% (w/v) 3-diaminobenzinidine (DAB; pH 3.8) for 1 h and subsequently rinsed with deionized water. To evaluate the presence of O , roots were stained with 2 mM nitroblue tetrozolium (NBT) in 20 mM phosphate-buffered saline (PBS; pH 6.8) for 15 min and subsequently rinsed with deionized water. DAB- or NBT-stained roots were visually observed using an Olympus FSX100 Bio-imaging navigator (Olympus America, Central Valley, PA) and pictures were captured using bright-field single-shot mode at 4.2x magnification.
8
Histochemical Detection of Reactive Oxygen
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Histochemical staining for the presence of hydrogen peroxide and superoxide was performed following 24 d stress treatment, using procedures described in Thordal-Christensen et al. [43 ] and Dunand et al. [44 (link)], with slight modifications respectively. To evaluate the presence of hydrogen peroxide (H2O2), a subset of roots was stained with 1% (w/v) 3-diaminobenzinidine (DAB; pH 3.8) for 2 h and subsequently rinsed with deionized water. To evaluate the presence of superoxide (O2-), a subset of roots was stained with2 mM nitroblue tetrozolium (NBT) in 20 mM phosphate-buffered saline (PBS; pH 6.8) for 30 min and subsequently rinsed with deionized water. DAB or NBT-stained roots were observed visually using an Olympus FSX100 Bio-imaging navigator (Central Valley, PA) and pictures were captured using bright-field single-shot mode at 4.2x magnification.
9
Immunohistochemical Analysis of Tumor Markers
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Formalin-fixed and using paraffin embedded samples were sliced into 5-μm-thick sections. Then, sample sections were immunostained using primary antibodies Ki67, Ras, cleaved caspase-3 and ARHI at 4 °C overnight. Then, applying secondary antibodies at 37 °C for 30 min. Next, samples were visualized according to manufacturer’s protocol and using a diaminobenzidine (DAB) substrate kit for 10 min. After intensive washing, samples were counterstained with hematoxylin, dehydrated and coverslipped. Obtaining pictures by using FSX100 Bio Imaging Navigator (Olympus, Japan).
10
Histological and Immunohistochemical Analysis
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Tissue samples were fixed in 4% paraformaldehyde, dehydrated in graded ethanol, embedded in paraffin and cut into 5 μm thick sections. H&E and Masson trichrome staining were used to detect histological changes and collagen deposition. For immunohistochemical staining, sections were deparaffinized and immersed in 3% H2O2 for 15 min at 37°C to remove terminal peroxidase activity, and blocked with 5% BSA in PBS for 1 h to eliminate nonspecific binding. The slides were then incubated overnight at 4°C with primary antibodies against α-SMA and Smad2. The next day, slides were incubated with the PV6000 Histostain™ kit (ZSGB, Beijing, China) and stained with diaminobenzidine (ZSGB, Beijing, China). Images were taken with an FSX100 Bioimaging Navigator (Olympus, Tokyo, Japan).
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