SVF preadipocytes were isolated from SAT and VAT of 6-week male WT, FGF21KO and Klb AdipoKO mice as in flow cytometry experiment. Cells were grown to confluency and differentiated in vitro over an 8-day period. Differentiation was induced with 2-day treatment of high glucose DMEM containing 10% FBS, 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone, 2 μM rosiglitazone and 10 μg ml−1 insulin, and with 6-day treatment of high glucose DMEM containing 10% FBS and 10 μg ml−1 insulin. Media was replaced every other day. Cells were treated with 200 ng ml−1 or 400 ng ml−1 rmFGF21. Differentiated cells were collected for measuring related gene expression or Oil Red O staining. Stained cells were visualized with an Olympus biological microscope BX41, and images were captured with an Olympus DP72 color digital camera.
Dp72 color digital camera
Manufactured by Olympus
Sourced in Japan
The DP72 is a color digital camera designed for microscopy applications. It features a high-resolution sensor and provides accurate color reproduction for capturing detailed images of specimens under a microscope. The camera is compatible with a range of Olympus microscopes and can be easily integrated into a microscopy workflow.
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Lab products found in correlation
Biological microscope bx41, by Olympus (5 mentions)
Oil red o, by Merck Group (2 mentions)
Horseradish peroxidase conjugated secondary antibody against rabbit igg, by Cell Signaling Technology (2 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Ab90395, by Abcam (1 mentions)
Ab32084, by Abcam (1 mentions)
Ab58655, by Abcam (1 mentions)
Ab126820, by Abcam (1 mentions)
A9434, by Merck Group (1 mentions)
Ht5011, by Merck Group (1 mentions)
Ab22552, by Abcam (1 mentions)
A10667, by Thermo Fisher Scientific (1 mentions)
H3570, by Thermo Fisher Scientific (1 mentions)
A3059, by Merck Group (1 mentions)
A11036, by Thermo Fisher Scientific (1 mentions)
Bx41 fluorescence microscope, by Olympus (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Merck Group (1 mentions)
Formalin solution, by Merck Group (1 mentions)
Hematoxylin eosin, by Merck Group (1 mentions)
7 protocols using dp72 color digital camera
1
FGF21 Regulates Adipocyte Differentiation
2
Histological Analysis of Adipose Tissue
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Tissues were cut into small pieces and fixed in 10% neutral buffered formalin solution for 48 h before transferring to 75% ethanol for long-term storage at 4°C. The paraffin blocks were prepared for sectioning at 5 μm thickness. The tissue sections were stained with H&E for examination. The size of adipocytes was measured randomly from ten fields and calculated as average cross-sectional area using the ImageJ software.
(Version 1.51, NIH, USA). For evaluation of lipid accumulation, frozen tissues embedded in Tissue-Tek OCT compound (Sakura® Finetek, CA, USA) were sectioned at 10 μm thickness and stained with Oil Red O (Sigma-Aldrich). All images were examined and captured using an Olympus biological microscope BX41, equipped with a DP72 color digital camera (Olympus, Tokyo, Japan).
(Version 1.51, NIH, USA). For evaluation of lipid accumulation, frozen tissues embedded in Tissue-Tek OCT compound (Sakura® Finetek, CA, USA) were sectioned at 10 μm thickness and stained with Oil Red O (Sigma-Aldrich). All images were examined and captured using an Olympus biological microscope BX41, equipped with a DP72 color digital camera (Olympus, Tokyo, Japan).
3
Histological Evaluation of NAFLD
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Tissues were cut into small pieces and fixed in 10% formalin solution for 48 h before transferring to 75% ethanol for long-term storage at 4 °C. The paraffin blocks were prepared for sectioning at 5μm thickness. The tissue slides were stained with hematoxylin and eosin (H&E) solution. Lesions of liver were evaluated with NASH CRN Scoring System. The grading for steatosis included 0: <5%; 1: 5–33%; 2: 34–66%; and 3: >66%. For lobular inflammation, the grades were 0: none; 1: <2 foci/20× field; 2: 2–4 foci/20× field; 3: >4 foci/20× field. The scores for hepatocellular ballooning were 0: none; 1: mild, few; 2: moderate-marked, many. The NASH CRN scores ranging from 0 to 8 were calculated as the sum of steatosis score (0–3), lobular inflammation score (0–3), and hepatocellular ballooning score (0–2)72 (link). After H&E staining of the adipose tissue sections, the size of adipocytes was measured and calculated using Image J software (Version 1.51, NIH, USA). Ten fields were randomly chosen and the size of adipocytes is presented as average cross-sectional area. Frozen liver tissues were embedded in Tissue-Tek OCT compound (Sakura® Finetek, CA, U.S.A.), sectioned at 5 μm and then stained with Oil Red O (Sigma-Aldrich) for 10 min. All slides were examined under Olympus biological microscope BX41, and images were captured using an Olympus DP72 color digital camera.
4
Immunofluorescence staining of cultured cells
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Cultured cell samples were carefully rinsed with PBS (Gibco, 21600-10), fixed with formalin solution (Sigma-Aldrich, HT5011, Madrid, Spain) for 20 min at room temperature, and rinsed again twice with PBS. The aldehyde groups were blocked with ammonium chloride (Sigma-Aldrich, A9434) 50 mM in PBS for 20 min. The samples were permeabilized with saponin (Sigma-Aldrich, 47036) 0.1% m/v in bovine serum albumin (BSA) (Sigma-Aldrich, A3059) 1% m/v in PBS for 10 min.
The samples were stained with the corresponding primary antibodies against paxillin (Abcam, ab32084, Cambridge, United Kingdom), scleraxis (Abcam, ab58655), collagen I (Abcam, ab90395), osterix (Abcam, ab22552), and alkaline phosphatase (Abcam, ab126820) in BSA 1% m/v PBS for 2 h at room temperature, then with the corresponding secondary fluorophore-conjugated antibodies anti-rabbit Alexa 568 (LifeTech, A11036, Madrid, Spain) and anti-mouse Alexa 488 (LifeTech, A10667) in BSA 1% m/v in PBS for 2 h. CytoPainter 488 (Abcam, ab176753) and Hoechst 33342 (Invitrogen, H3570) were used for actin filaments and cell nuclei staining. The samples were mounted with coverslips in fluoromount mounting medium (Sigma-Aldrich, HT5011).
The samples were imaged at a Nikon E600 upright manual microscope with a 40X/0.75 NA objective and an Olympus DP72 color digital camera. At least three representative images were taken of each sample.
The samples were stained with the corresponding primary antibodies against paxillin (Abcam, ab32084, Cambridge, United Kingdom), scleraxis (Abcam, ab58655), collagen I (Abcam, ab90395), osterix (Abcam, ab22552), and alkaline phosphatase (Abcam, ab126820) in BSA 1% m/v PBS for 2 h at room temperature, then with the corresponding secondary fluorophore-conjugated antibodies anti-rabbit Alexa 568 (LifeTech, A11036, Madrid, Spain) and anti-mouse Alexa 488 (LifeTech, A10667) in BSA 1% m/v in PBS for 2 h. CytoPainter 488 (Abcam, ab176753) and Hoechst 33342 (Invitrogen, H3570) were used for actin filaments and cell nuclei staining. The samples were mounted with coverslips in fluoromount mounting medium (Sigma-Aldrich, HT5011).
The samples were imaged at a Nikon E600 upright manual microscope with a 40X/0.75 NA objective and an Olympus DP72 color digital camera. At least three representative images were taken of each sample.
5
Apoptosis Quantification in H9C2 Cardiomyocytes
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H9C2 cardiomyocytes were cultured directly on coverslips. After the treatment, the cardiomyocytes' apoptosis was determined by TUNEL staining using an In Situ Cell Death Detection Kit (Cat. number 11684817910, Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer's instructions. Images were taken using an Olympus BX41 fluorescence microscope equipped with an Olympus DP72 color digital camera (Olympus, Tokyo, Japan). Five photos (magnification ×200) were taken randomly for each sample. Image quantification was presented as percentage of TUNEL-positive cells among the total number of cells as described [31 (link)].
6
Immunohistochemical Analysis of Liver FGF21
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Liver specimens collected before or 2 hours post-implantation were fixed in 10% formalin solution (Sigma-Aldrich, USA); and paraffin-embedded liver sections were stained with hematoxylin-eosin (Sigma-Aldrich, USA). For FGF21 immunohistochemical staining, deparaffinized and dehydrated liver sections were incubated with an affinity-purified rabbit antibody against FGF21 (Antibody and Immunoassay Services, Hong Kong, China) in a solution containing 3% bovine serum albumin overnight at 4 °C, followed by the reaction with a horseradish peroxidase-conjugated secondary antibody against rabbit IgG (Cell Signaling Technology, USA) and 3,3′-Diaminobenzidine tetrahydrochloride (Sigma-Aldrich, USA). Immuno-stained slides were visualized with an Olympus biological microscope BX41, and images were captured with an Olympus DP72 color digital camera.
7
Immunohistochemical and ELISA analysis of adiponectin
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Adipose tissues were fixed in 10% formalin solution for 24 hr, embedded in paraffin, and sectioned at 5 mm. Deparaffinized and dehydrated sections were incubated with affinity-purified rabbit antibody against mouse adiponectin (AIS, HKU), rabbit antibody against mouse UCP1 (Abcam) in PBS containing 3% BSA overnight at 4 C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody against rabbit IgG (Cell Signaling Technology), and developed by SIGMAFAST DAB (Sigma). For immunocytochemical staining, bone marrow-derived macrophages were fixed in ice-cold 4% formaldehyde for 10 min. The cells were then incubated with antibody against Ki67 (Abcam) or adiponectin in PBS containing 3% BSA overnight at 4 C, washed and incubated with fluorochrome-conjugated secondary antibodies (Alexa Fluor 555 anti-rabbit, Molecular Probes), and diluted in PBS for 1 hr at room temperature at dark. Slides were counterstained with DAPI. Tissue sections and cell slides were visualized with an Olympus biological microscope BX41, and images were captured with an Olympus DP72 color digital camera. Adiponectin levels in serum were measured by mouse adiponectin ELISA kit (AIS, HKU).
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