Histology and immunohistochemistry (IHC) were carried out on paraformaldehyde-fixed histological IP and NU tissue sections (n = 8 and 9 respectively). Haematoxylin and eosin staining was performed according to standard protocols. IHC staining was carried out using EnVision peroxidase/DAB+ detection system (Dako, Stockport, UK) and specific anti-human antibodies (Supplementary Table S1 ). In cases where antigen retrieval was required, this was performed by heating sections in boiling 10 nM citrate buffer (pH 6.0) in a 900 W microwave oven for 10 min. All images were captured using an Eclipse E1000 light microscope (Nikon). For measurement of average blood vessel size, up to six photomicrographs (dependent on tissue size) were taken at 200× magnification following CD31 labelling. Measurement of blood vessel areas was then performed blinded using ImageJ free access image processing and analysis software available from http://rsb.info.nih.gov/ij/ . The number of vessels was counted in at least 20 different random fields of each experimental group, as previously described [48 (link)]. Analysis of CD45, stromal-derived factor 1 (SDF1), vascular endothelial growth factor 1 (VEGF), and bone morphogenetic protein 2 (BMP-2) expression was performed using NIS elements BR (Nikon Instruments Inc., Melville, USA ) image analysis software.
Envision detection system peroxidase dab
Manufactured by Agilent Technologies
Sourced in Denmark
The EnVision Detection System Peroxidase/DAB is a laboratory equipment product designed for use in immunohistochemistry and immunocytochemistry applications. It is a detection system that utilizes peroxidase-based labeling and 3,3'-diaminobenzidine (DAB) as the chromogenic substrate.
Automatically generated - may contain errors
Lab products found in correlation
Protein blocking solution, by Agilent Technologies (2 mentions)
Sodium citrate ph 6, by Merck Group (2 mentions)
Eclipse e1000 light microscope, by Nikon (1 mentions)
Nis elements br, by Nikon (1 mentions)
Oil red o, by Merck Group (1 mentions)
Sab3500340, by Merck Group (1 mentions)
Envision reagent, by Agilent Technologies (1 mentions)
Dako real peroxidase blocking solution, by Agilent Technologies (1 mentions)
Ultravision quanto detection system hrp dab, by Thermo Fisher Scientific (1 mentions)
Direct red 80, by Merck Group (1 mentions)
Anti f4 80 antibody, by Cell Signaling Technology (1 mentions)
Fast green fcf, by Merck Group (1 mentions)
8 protocols using envision detection system peroxidase dab
1
Histological and Immunohistochemical Analysis of Tissue
2
Immunohistochemical Analysis of Placental Proteins
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Paraffin-embedded placental tissue biopsies were cut into 5 µm sections, deparaffinized with xylene, and then gradually rehydrated with decreasing concentrations of ethyl alcohol. Boiled 10 mM citrate buffer (pH 6.0) was used for antigen retrieval and endogenous peroxidase activity was blocked in 3% hydrogen peroxidase prepared in PBS. After blocking further with 10% rabbit serum, sections were probed overnight at 4 degree Celsius with either rabbit anti-LEPTIN antibody (1:100, Proteintech, USA) or rabbit anti-ARMS2 antibody (1:200, Novus, USA). Specific protein expression was visualized using the EnVision Peroxidase/DAB detection system (Dako, Denmark) according to the manufacturer's instructions. Negative control sections were treated similarly, except the primary antibody, was excluded.
3
Evaluation of MORC2 Expression in Tumor Cells
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The expression of MORC2 on tumor cells was evaluated through the additional paraffin-embedded biopsy samples. Immunohistochemistry (IHC) staining was carried out using EnVision Detection System Peroxidase/DAB (DAKO, Santa Clara, CA) following the manufacturer’s recommendations. The primary antibody against human MORC2 (Novus, #NBP1-89295) was diluted at dilution of 1:100 and then incubated at 4°C overnight in a humidified container. Positive and negative controls were used for each run of staining. Interpretation of the IHC results was performed by 2 independent pathologists who were blinded to the clinicopathological information. Slides were evaluated using light microscopy and a standard semiquantitative immunoreactivity score. Whether there is cytoplasmic staining or not, MORC2 was considered to be positive as long as there is nuclear staining, either strong or weak. The percentage of MORC2 was evaluated on all of the neoplastic cells in each biopsy samples and scored as 0% to 100%. The intensity of nucleus staining was recorded through the semi-quantitative methods as absent (0), weak (1), moderate (2), and strong (3). The median value of MORC2 expression was calculated and the specimens were classified into 2 groups: MORC2 expression levels below and equal to the median value as “low-MORC2” and above the median value as “high-MORC2,” respectively.
4
Histological Analysis of Liver Oxidative Stress
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Tissue specimens were fixed in 10% formalin for 12 to 24 h, dehydrated, and paraffin embedded. Liver sections were stained with H&E following standard protocols. For immunohistochemistry, sections were subjected to antigen retrieval by boiling the slides in sodium citrate pH 6 (Sigma Aldrich) for 15 min and then permeabilized in phosphate-buffered saline (PBS) with 0.25% TritonX-100 for 5 min. Subsequently, after 10 min incubation at room temperature in protein blocking solution (Dako, Glostrup, Denmark), sections were incubated at 4 °C for 48 hours with the anti-8-Hydroxyguanosine antibody (LifeSpan Bioscience Inc, Seattle, Washington, USA). Sections were washed in PBS for 15 min and incubated for 25 min at room temperature with DAKO real EnVision detection system Peroxidase/DAB + (Dako) according to manufacturer’s instruction. Coverslips were mounted with Permount and evaluated under a light microscope. To evaluate lipid accumulation, serial 4.5 μm cryosections from liver specimens embedded in OCT compound (Tissue-Tek Sakura, Torrance, CA) were stained with Oil Red O (Sigma-Aldrich) and hematoxylin to counterstain nuclei.
5
GFAT1 Immunohistochemistry Scoring Protocol
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Immunohistochemistry was performed using a two-step procedure following the protocol recommended by Dako REAL™ EnVision™ Detection System, Peroxidase/DAB+ (Cat# K5007, Dako, Denmark). To ensure antibody specificity, control slides were incubated either in the absence of primary antibody or with a nonspecific IgG antibody. Positive brownish cytoplasm immunostaining in more than 5% of tumor cells was the criterion for immunostaining positivity for GFAT1. Slides were assessed by an experienced and independent pathologist blinded to the patient's status. To obtain an IHC score that takes into account the IHC signal intensity and the frequency of positive cells, a composite expression scores (CES) with full range from 0 to 12 was generated as described in our previous report [19 (link)].
6
Immunohistochemistry of PTK7 Protein
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The 4-µm thick slices were baked at 72°C for 1 h, and then dewaxed and rehydrated through xylene and alcohol with graded concentrations. Hydrogen peroxide (3%) was used to eliminate activity of endogenous peroxidase for 15 min. After PBS washing 3 times, the antigen retrieval was performed in a pressure cooker with EDTA (pH 8.0) (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). After cooling to room temperature, the sections were blocked with 5% skimmed milk at room temperature for 1 h, followed by incubation with primary rabbit polyclonal anti-PTK7 antibody (1:7,500, SAB3500340; Sigma-Aldrich, St. Louis, MO, USA) at 4°C overnight. The ready-to-use EnVision™ reagent (EnVision™ detection system peroxidase/DAB, rabbit/mouse; Dako, Glostrup, Denmark) was then used to bind the primary antibody. The 3,3′-diaminobenzidine tetrahydrochloride kit (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. was used to develop substrates. After being counterstained with hematoxylin, the sections were dehydrated with graded alcohol and xylene.
7
Immunohistochemical Staining of Vascular and Lymphatic Markers
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All immunohistochemical reactions were conducted with formalin-fixed and paraffin-embedded samples.
CD31, D2-40: After deparaffination heat-induced epitope retrieval was performed in Tris-EDTA buffer pH 9,0 for 20 min. using a vegetable steamer. Non-specific binding was blocked by Dako REAL™ Peroxidase-Blocking Solution (Dako, Hamburg, Germany) prior to incubation with the primary antibody. For the immunohistochemical staining procedure DAKO REAL™EnVision™Detection System, Peroxidase/DAB+, Rabbit/Mouse was utilized following the provider’s instructions. The primary antibodies mouse monoclonal CD31 (Dako, Hamburg, Germany) and mouse monoclonal D2-40 (Signet, Dedham, MA, USA) were applied at a dilution of 1:50 for 1 h at room temperature. Sections were counterstained with Mayer’s haematoxylin.
sm-actin: After deparaffination the primary antibody mouse monoclonal sm-actin (Zytomed Systems, Berlin, Germany) was added at a dilution of 1:800 for 45 minutes at room temperature. UltraVision Quanto Detection System HRP DAB (Thermo Scientific, Fremont, CA, USA) was used for the staining procedure following the provider’s instructions. Sections were counterstained with Mayer’s haematoxylin.
CD31, D2-40: After deparaffination heat-induced epitope retrieval was performed in Tris-EDTA buffer pH 9,0 for 20 min. using a vegetable steamer. Non-specific binding was blocked by Dako REAL™ Peroxidase-Blocking Solution (Dako, Hamburg, Germany) prior to incubation with the primary antibody. For the immunohistochemical staining procedure DAKO REAL™EnVision™Detection System, Peroxidase/DAB+, Rabbit/Mouse was utilized following the provider’s instructions. The primary antibodies mouse monoclonal CD31 (Dako, Hamburg, Germany) and mouse monoclonal D2-40 (Signet, Dedham, MA, USA) were applied at a dilution of 1:50 for 1 h at room temperature. Sections were counterstained with Mayer’s haematoxylin.
sm-actin: After deparaffination the primary antibody mouse monoclonal sm-actin (Zytomed Systems, Berlin, Germany) was added at a dilution of 1:800 for 45 minutes at room temperature. UltraVision Quanto Detection System HRP DAB (Thermo Scientific, Fremont, CA, USA) was used for the staining procedure following the provider’s instructions. Sections were counterstained with Mayer’s haematoxylin.
8
Liver Histopathological Analysis in NAFLD
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Tissue samples were fixed in 4% formalin for 24 h, dehydrated, and paraffin‐embedded. Liver sections were stained with hematoxylin and eosin (H&E) following standard protocols. Steatosis score was calculated according to Kleiner et al.[17 ] For immunohistochemistry, sections were subjected to antigen retrieval by boiling the slides in sodium citrate pH 6 (Sigma Aldrich) for 15 min and then permeabilized in phosphate‐buffered saline (PBS) with 0.25% TritonX‐100 for 5 min. Subsequently, after 10‐min incubation at room temperature in protein‐blocking solution (Dako), sections were incubated at 4°C for 24 h with the anti‐F4/80 antibody (#70076; Cell Signaling Technology). Sections were washed in PBS for 15 min and incubated for 25 min at room temperature with the Dako real EnVision detection system (Peroxidase/DAB+) according to the manufacturer's instructions. For hepatic fibrosis evaluation, 4‐μm‐thick sections were stained with Direct Red 80 and Fast Green FCF (Sigma‐Aldrich). Image processing was performed using ImageJ (National Institutes of Health) software.
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