Fresh biopsy specimens from liver and adipose tissue were fixed in 10% neutral-buffered formalin for 24 hours, embedded in paraffin, sectioned using a Leica SM2010 R Sliding microtome (Shanghai, China) and stained with hematoxylin-eosin (HE) or Oil red O to assess histopathological features. For immunohistochemistry staining, adipose tissue sections were immunostained with anti-PGC-1α (1:200, Abcam) antibody using a DAB Substrate Kit (MXB Biotechnologies, Fuzhou, China) and counterstained with hematoxylin. Stained areas were viewed and imaged using standard microscopy (Nikon, Shanghai, China).
Dab substrate kit
Manufactured by Beijing Strong Biotechnologies
Sourced in China
The DAB substrate kit is a reagent system used in immunohistochemistry (IHC) and immunocytochemistry (ICC) applications. It provides a chromogenic substrate for the visualization of target antigens in fixed tissue sections or cell samples. The kit contains the necessary components to perform the substrate reaction, enabling the detection and localization of specific proteins or other biomolecules of interest.
Automatically generated - may contain errors
Lab products found in correlation
Sm2010r sliding microtome, by Leica (1 mentions)
Anti pgc 1α, by Abcam (1 mentions)
Pannoramic midi scanner, by 3DHISTECH (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Collagenase type 2, by Merck Group (1 mentions)
Goat anti rabbit igg, by Affinity Biosciences (1 mentions)
Anti col2a1, by Abcam (1 mentions)
Dp72 microscope, by Olympus (1 mentions)
Bcip nbt alkaline phosphatase color development kit, by Beyotime (1 mentions)
Anti b220, by BD (1 mentions)
Anti bcl6, by BioLegend (1 mentions)
Maxvision kit, by Beijing Strong Biotechnologies (1 mentions)
7 protocols using dab substrate kit
1
Histopathological Analysis of Liver and Adipose Tissue
2
Immunohistochemical Staining of HNSCC Samples
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Immunohistochemical staining was conducted as previously described [59 (link)]. Briefly, human and mouse HNSCC samples were paraffin embedded and sectioned at 4 μm. Following xylene deparaffinization and gradient ethanol soaking rehydration, the sections were subjected to high-temperature antigen retrieval with a citrate antigen retrieval solution and endogenous peroxidase inhibition with 3% hydrogen peroxide. Then, the non-specific binding was blocked using 5% goat serum (Mxb Biotechnologies, Fuzhou, China, KIT-9710). The processed sections were then incubated with primary antibodies (Table S2 ) at 4 °C overnight, washed with PBS, and incubated with horseradish peroxidase-conjugated secondary antibodies (Mxb Biotechnologies, KIT-9710) at 37 °C for 30 min. A DAB substrate kit (Mxb Biotechnologies, DAB-0031) and hematoxylin were used for staining. Images were visualized using a Pannoramic MIDI scanner (3DHISTECH) and quantified using the Aperio ScanScope software (v11.1.2.752, Aperio, San Diego, CA, USA). IgG was used as a negative control.
3
Isolation and Characterization of Rat Condylar Chondrocytes
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Supported by the Model Animal Research Center of Kunming Medical University, condylar cartilage tissues (Figure 1(a) ) of 2-day-old SD (Sprague Dawley) rats (male, 5 g) were cut and digested with 0.25% trypsin (Gibco Invitrogen, USA) at 37°C for 20 min, then with 0.2% collagenase type II (Sigma Aldrich, USA) at 37°C for 1 h [27 (link)]. The cells were incubated in the DMEM/high glucose medium (BI, Israel) containing 10% fetal bovine serum (Gibco Invitrogen) (Figure 1(b) ). MCCs (2 × 104 cells/ml) were fixed with 4% paraformaldehyde when they reached 70% confluence. Immunocytochemistry was performed as follows: cells at the 1st passage were incubated with 0.4% Triton X-100 for 30 min, blocked with 3% H2O2 for 15 min, and incubated with 10% goat serum for 30 min, and primary antibodies (anti-Col2A1, 1 : 50 dilution; Abcam, USA) were added at 4°C overnight. Secondary antibodies (Goat Anti-Rabbit IgG, 1 : 200 dilution; Affinity Biosciences, USA) were added for 1 h, stained with the DAB substrate kit (MXB, Fuzhou, China), and restained with hematoxylin, and the slides were sealed after gradient ethanol dehydration and air-drying. Toluidine blue staining (Solarbio, Beijing, China) was performed for 30 min, rinsing with running water and air-drying.
4
Quantifying Cell Proliferation and Apoptosis
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To assess the cell proliferation, the immunohistostaining with antibody against Ki67 (511390; ZEN Bio Inc., China) was performed on the 10-μm-thick paraffin sections. The HRP-conjugated anti-rabbit/mouse IgG (MXB Biotechnologies Inc., Fujian, China) was used as the secondary antibody. The color was developed with the DAB substrate kit (MXB Biotechnologies Inc., Fujian, China), following the manufacturer’s instruction. Hematoxylin was used for counter-staining. The TUNEL assay was also performed on 10-μm-thick paraffin sections with the In Situ Cell Death Detection Kit, POD (11684817910, Roche Diagnostics Corporation, Indianapolis). The procedure for apoptosis detection followed the manufacturer’s instructions. The sections were counter-stained with DAPI and observed by the Olympus DP72 microscope. The percentages of cell proliferation were determined by the numbers of Ki67-positive nuclear to the numbers of the total nuclei. The densities of cell apoptosis were calculated by TUNEL fluorescence signal in a fixed area. To evaluate whether the differences in cell proliferation and death were significant, Student’s t-tests were used to evaluate the pairs of the WT and Osr2-creKI;pMes-Noggin soft palates. The results were presented as mean ± SD of at least three pairs of samples. When the p-value was less than 0.05, the difference was considered to be statistically significant.
5
Comprehensive Liver Histopathological Assessment
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Liver tissue was fixed in 4% paraformaldehyde neutral solution for 24 hours, the liver tissues were divided into two parts, one part was paraffin embedded, 4 um thick sections were cut and stained with hematoxylin-eosin or Masson staining to assess histological features. For Oil Red O staining, frozen liver sections (5μm) were fixed with fixative for 15 minutes and then stained with freshly prepared Oil Red O solution for 15 minutes. The sections were subsequently rinsed again with 60% isopropanol and finally the nuclei were stained with hematoxylin. The staining was examined under the microscope. For immunohistochemistry, paraffin sections (4um) of liver were immunostained with anti-Dio1 (1:200, 11790-1-AP, Proteintech) using a DAB substrate kit (MXB Biotechnologies, Fuzhou, China) and restained with hematoxylin. Images were captured with a Motic VM1 microscope (McAudi, Hong Kong, China). Digital images were processed with Adobe Photoshop (Adobe, San Jose, CA, USA) and quantitatively analyzed with Image J software (National Institutes of Health, Bethesda, MD, USA).
The histological features of the liver were scored using the NAFLD activity scoring (NAS) system (19 ) based on HE staining. NAS consists of three aspects: steatosis (0-3), lobular inflammation (0-3), hepatocellular enlargement (0-2).
The histological features of the liver were scored using the NAFLD activity scoring (NAS) system (19 ) based on HE staining. NAS consists of three aspects: steatosis (0-3), lobular inflammation (0-3), hepatocellular enlargement (0-2).
6
Immunohistochemical Identification of Germinal Centers
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Spleen and lymph nodes were fixed in formalin prior to sectioning. Four micron thick sections were cut and stained. For GC identification, 4 mM LNs and spleen sections were stained with anti-Bcl6 (Biolegend, diluted 1:400) and anti-B220 (BD Bioscience, diluted 1:10) antibodies. Secondary antibodies, anti-mouse IgG-HRP (MXB biotechnologies) and Anti-Rat IgG-AP (BETHYL), were used to amplify the detection signal. A DAB Substrate Kit (MXB biotechnologies) and BCIP/NBT Alkaline phosphatase Color Development Kit (Beyotime Biotechnology) were used according to the manufacturer's protocol.
7
Immunohistochemistry with Anti-Fascin Antibody
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Mouse anti-human Fascin_1 monoclonal antibody (SPM133) for immuno-histochemistry was purchased (Santa Cruz, USA). The immuno-histochemical test kit (MaxVision kit) and DAB substrate kit were purchased from MXB Biotechnologies Co.
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