Heart samples were collected and fixed overnight in 4% paraformaldehyde (BL539A, Biosharp, Hefei, Anhui, China), followed by routine dehydration and sectioning (5 μm). Sections were blocked with 3% hydrogen peroxide, antigen repaired with citrate buffer (P0083, Beyotime, Shanghai, China), permeabilized with 0.3% Triton-100 (ST795, Beyotime, Shanghai, China), blocked with 5% BSA (A1933, Sigma-Aldrich, St. Louis, MO), incubated with primary antibody overnight at 4 °C and incubated with HRP-labeled secondary antibody for 30 min at 37 °C. Visualization was performed under the microscope (DFC700T, Leica, Germany) with DAB Horseradish Peroxidase Color Development Kit (P0203, Beyotime, Shanghai, China). Finally, the sections were sealed with neutral balsam fixative (G8590, Solarbio, Beijing, China). The positive cell number was counted from 4–5 fields per sample with the ImageJ software program (v.1.45, National Institutes of Health, Bethesda, MD, USA) and the mean density was quantified from 4–5 fields per sample with the Image-pro plus software program (v.6.0, Media Cybernetics, Rockville, MD, USA). The antibodies are listed in Supplementary Table 1 .
Dfc700t
Manufactured by Leica camera
Sourced in Germany
The DFC700T is a high-performance digital camera designed for scientific and industrial applications. It features a high-resolution CMOS sensor and provides advanced image capture capabilities for a variety of microscopy and imaging needs. The DFC700T is a compact and versatile solution for capturing detailed and accurate images in various laboratory and research settings.
Automatically generated - may contain errors
Lab products found in correlation
P0083, by Beyotime (2 mentions)
A1933, by Merck Group (2 mentions)
St795, by Beyotime (2 mentions)
Bl539a, by Biosharp (2 mentions)
Dm2500 compound microscope, by Leica camera (2 mentions)
Tunel kit, by Merck Group (2 mentions)
Dab horseradish peroxidase color development kit, by Beyotime (1 mentions)
G8590, by Solarbio (1 mentions)
Image pro plus software program, by Media Cybernetics (1 mentions)
Oil red o, by Merck Group (1 mentions)
Tunel kit, by Beyotime (1 mentions)
Wga fitc, by Merck Group (1 mentions)
Andor spinning disk confocal microscope, by Oxford Instruments (1 mentions)
12 protocols using dfc700t
1
Immunohistochemical Analysis of Heart Samples
2
Oil Red O Staining of Frozen Tissue
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3
Heart Tissue Immunofluorescence Staining
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Heart samples were collected and fixed in 4% paraformaldehyde (BL539A, Biosharp, Hefei, China) overnight followed by conventional dehydration and slicing (5 μm). The heart samples sections were blocked with 3% hydrogen peroxide and then performed at 95 °C for 10 min using citrate buffer (P0083, Beyotime, Shanghai, China), 0.3% Triton-100 (ST795, Beyotime, Shanghai, China) was used for permeabilization and then the blocking step was carried out using the 5% BSA (A1933, Sigma-Aldrich, St. Louis, MO). After overnight incubation of the primary antibody at 4 °C, a secondary antibody was applied to the sections at 37 °C for 90 min. All the immune-fluorescence images were captured by a fluorescence microscope (DFC700T, Leica, Germany). The antibody is listed in Supplementary Table 1 .
4
Immunohistochemistry Protocol for Brain Sections
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Immunohistochemistry (IHC) was performed as described previously (McCall et al., 2017). Briefly, mice were euthanized with sodium pentobarbital and transcardially perfused with 4% paraformaldehyde (PFA), post-fixed for 1-3 days in 4% PFA, and then cryo-protected in 30% sucrose. Brain sections (30-100μm) were collected and kept in 0.1M PB at 4°C until IHC. Sections were washed 3 times for 10min in PBS, then 1h in blocking buffer (0.5% Triton X-100 and 5% goat serum in 0.1M PBS) at room temperature, followed by overnight incubation with primary antibody () at 4°C. Next, the sections were again washed 3 times for 10min in PBS, incubated for 1h in Alexa Fluor secondary antibodies (1:1000) at room temperature, and washed 3 times for 10min in PBS. Then, the sections were mounted with VectaShield Vibrance Hardset mounting medium (Vector Laboratories) with DAPI, and coverslips placed. Images were acquired on a epifluorescent microscope (Leica DFC700T).
5
Histopathological Analysis of Kidney and Liver
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The kidney and liver tissues were fixed in 4% paraformaldehyde (pH 7.4) overnight for histological analysis, embedded in paraffin, and serially sectioned at 3.5 μm. Standard hematoxylin and eosin staining, Masson’s trichrome stain, Sirus red staining and PAS staining were used. GBM thickness was determined by PASM staining. All the histological staining images were captured by a microscope (DFC700T, Leica, Germany).
6
Apoptosis Detection via TUNEL Assay
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Sections were deparaffinized and hydrated in xylene and gradient concentrations of ethanol, then incubated in proteinase K at room temperature for 30 min and stained with TUNEL kit (C1091, Beyotime Institute of Biotechnology, China). TUNEL assay was then performed according to the instructions by the manufacturer. All the histological staining images were captured by a microscope (DFC700T, Leica, Germany).
7
Embryo Fixation and In Situ Hybridization
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Fixation and preparation of embryos for tissue sectioning and in situ hybridization were performed as previously described (50 (link), 51 (link)). For in situ experiments, cRNA probes were generated by in vitro reverse transcription of cDNAs. Digoxigenin- (dig-) UTPs were incorporated into probes for detection with anti-dig antibodies conjugated to alkaline phosphatase and visualized with NBT-BCIP substrate. Images were captured using a Leica DM2500 compound microscope with a Leica DFC700T camera system. In situs were viewed and photographed using Nomarski (differential interference contrast) optics and brightfield optics.
8
Apoptosis Quantification in Diabetic Hearts
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The hearts were fixed in 4% paraformaldehyde (pH 7.4) overnight, embedded in paraffin, and serially sectioned at 5 μm. Sections were deparaffinized and hydrated in xylene and gradient concentrations of ethanol, then incubated in proteinase K at room temperature for 30 min and stained with TUNEL kit (Sigma-Aldrich, St. Louis, MO, USA). Label solution was used instead of TUNEL reagent in the negative control group. All the images were captured by a fluorescence microscope (DFC700T, Leica, Germany). Cells that were positive for TUNEL staining and aligned with DAPI staining were considered apoptotic cells and counted. The number of apoptotic cells in the diabetic group and control group was calculated. The data analysis was based on three individual mice per group.
9
Detecting Apoptosis in Tissue Sections
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Tissue sections were deparaffinized and hydrated in xylene and gradient concentrations of ethanol, then incubated in proteinase K at room temperature for 30 minutes and stained with TUNEL kit (Sigma‐Aldrich). Label solution was used instead of TUNEL reagent in the negative control group. All the images were captured by a fluorescence microscope (DFC700T, Leica). Cells that were positive for TUNEL staining and aligned with DAPI staining were considered apoptotic cells and counted.
10
Cardiomyocyte Area Quantification
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The cTNT was staining first following the method of Immunofluorescence, and then the sections were incubated with WGA-FITC (W11261, Sigma-Aldrich, St. Louis, MO) at 37 °C for 30 min. All the immune-fluorescence images were captured by a fluorescence microscope (DFC700T, Leica, Germany). The cardiomyocyte area was calculated from 4–5 fields per sample with the ImageJ software program (v.1.45, National Institutes of Health, Bethesda, MD, USA).
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