The presence of Lewy bodies in the substantia nigra and more importantly in the cingulum was verified with stained sections from the Netherlands brain bank (NBB). For those cases were no staining was available, we obtained paraffin sections from the NBB and performed a staining with an antibody directed against aggregated α-synuclein (anti-human α-synuclein 5G4, mouse monoclonal, analytikjena, Jena, Germany). After deparaffinization, antigen retrieval was performed by cooking in citrate buffer for 20 min and DAB staining was performed with the Envision Dual Link System-HRP DAB+ Kit (Agilent, Santa Clara, CA, USA) according to the manufacturer’s instructions. Briefly, the sections were blocked with Dual Endogenous Enzyme Block for 10 min and rinsed with PBS. Then, primary antibody was applied (diluted 1:500 in PBS + 1% BSA) for 40 min and again the slides were washed with PBS. Afterwards, labelled polymer was added for 30 min. After a further washing step, substrate was added for five minutes. Afterwards, the slides were washed in running tap water, counterstained in hemalaun (Dako/Agilent) and again rinsed in running tap water. Finally, the slides were dehydrated in increasing ethanol concentrations and xylol and mounted in entellan mounting medium (Merck).
Envision dual link system hrp dab kit
Manufactured by Agilent Technologies
Sourced in United States, Japan, United Kingdom
The Envision® + Dual Link System-HRP (DAB+) kit is a laboratory equipment product from Agilent Technologies. It is designed for immunohistochemical staining and visualization of target antigens in tissue samples. The kit includes reagents for a two-step detection system that utilizes a polymer-based horseradish peroxidase (HRP) conjugate.
Automatically generated - may contain errors
Lab products found in correlation
Target retrieval solution, by Agilent Technologies (2 mentions)
Entellan mounting medium, by Merck Group (1 mentions)
Umb 1, by Abcam (1 mentions)
All in one fluorescence microscope, by Keyence (1 mentions)
Xylene, by Merck Group (1 mentions)
Aperio digital pathology slide scanner, by Leica (1 mentions)
Mayer s hematoxylin, by ScyTek Laboratories (1 mentions)
3 3 diaminobenzidine dab substrate kit, by Abcam (1 mentions)
Dab substrate chromogen solution, by Agilent Technologies (1 mentions)
Ab5690, by Abcam (1 mentions)
Citrate buffer solution, by Merck Group (1 mentions)
Anti tgf β1 antibody, by Abcam (1 mentions)
Antibody diluent, by Agilent Technologies (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Bx50 fluorescence microscope, by Olympus (1 mentions)
S2031, by Agilent Technologies (1 mentions)
H0146, by Merck Group (1 mentions)
Hhs32, by Merck Group (1 mentions)
Zen blue software, by Zeiss (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
19 protocols using envision dual link system hrp dab kit
1
Immunohistochemical Detection of α-Synuclein
2
Immunohistochemical Analysis of Liver Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four-micron-thick sections of the formalin-fixed, paraffin-embedded tissue blocks of all the studied cases (including their paired paracancerous tissues) were prepared for immunohistochemistry targeted to GPC3, Arg-1 and HepPar-1. Immunohistochemistry was performed using the immunoperoxidase method. Positive control and negative control were set. In brief, sections were deparaffinized with xylene and rehydrated through a series of ethanol solutions. Heat-induced antigen retrieval was conducted in 0.1 mol/L citrate buffer (pH 6.0) in a microwave for 20 min. Endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 15 min. Pretreated sections were incubated with primary mouse monoclonal antibody against GPC3 (clone 7D11; 1:100 dilution; Darui Biotechnology, Guangzhou, China), HepPar-1 (clone OCH1E5; 1:200 dilution; DakoCytomation, Carpinteria, CA, USA) and Arg-1 (HPA003595; 1:200 dilution, Merck KGaA, Darmstadt, Germany) overnight at 4 °C. The reaction was detected with EnVision™ + Dual Link System-HRP (DAB) kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Sections were counter stained with hematoxylin for 15 sec before being checked under a microscope. For the negative control, phosphate-buffered saline was substituted for the primary antibody.
3
Immunohistochemical Evaluation of Somatostatin Receptor Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SST protein expression was evaluated in formalin-fixed paraffin-embedded tumor samples (5 µm-thick sections) using the Dako EnVision®+ Dual Link System-HRP (DAB+) kit (Dako/Agilent, Santa Clara, CA, USA) as previously reported [13 (link)]. To detect SST2 and SST5, 1:200 dilution of rabbit anti-SST2 (RRID: AB_2737601) and rabbit anti-SST5 (RRID: AB_10859946) monoclonal antibodies (UMB-1 and UMB-4, respectively, Abcam, Cambridge, UK) was used. Receptor staining on tumor samples was semiquantitatively scored by using an immunoreactivity scoring system (IRS). IRS is calculated by the product of the percentage of positive cells (4, >80%; 3, 51% to 80%; 2, 10% to 50%; 1, <10%; 0, 0%) and the intensity of the staining (3, strong; 2, moderate; 1, mild; 0, no staining), which results in IRS scores between 0 (no staining) and 12 (maximum staining) [13 (link)].
4
Immunohistochemical Staining and Mitotic Index Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue sections were fixed in 10% buffered formalin and embedded in paraffin. Tissue sections were routinely stained with hematoxylin and eosin. For IHC staining, tissue slides were deparaffinized in xylene and rehydrated in alcohol. Endogenous peroxidase was blocked with 3% hydrogen peroxide. Antigen retrieval was achieved using a hot water bath and 10 mM citric sodium buffer (pH 6.0). Sections were then incubated overnight at 4 °C with the indicated primary antibody. Antibody binding was detected with EnVisionTM Dual Link System-HRP DAB kit (K4010, Dako). Sections were then counterstained with hematoxylin. For negative controls, the primary antibody was excluded. The mitotic index was quantified by viewing and photographing 10 random HPFs for each tissue section on a Keyence All-In-One Fluorescence Microscope, using a 40× or 20× objective. For evaluation and scoring of immunohistochemical data, we randomly selected 10 fields within the tumor area under high-power magnification (×40) for evaluation. The investigators conducted blind counting for all quantification.
5
Immunohistochemistry protocol for tissue analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue sections were fixed in 10% neutral buffered formalin and embedded in paraffin. Tissue sections were routinely stained with hematoxylin and eosin. For immunohistochemistry staining, tissue slides were deparaffinized in xylene and rehydrated in alcohol. Endogenous peroxidase was blocked with 3% hydrogen peroxide. Antigen retrieval was performed by incubating the sections in boiling 10 mM sodium citrate pH 6.0 followed by incubation with the indicated primary antibody overnight at 4 °C. Antibody binding was detected with EnVisionTM Dual Link System-HRP DAB kit (K4010, Dako). Sections were then counterstained with hematoxylin. For negative controls, the primary antibody was excluded. For evaluation and scoring of immunohistochemical data, we randomly selected 10 fields within the tumor area under high power magnification (×40) for evaluation. The investigators conducted blind counting for all quantification.
6
Immunohistochemical Analysis of Mouse Tissues
7
Immunohistochemical Analysis of Tumor Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The paraffin-embedded tissue sections were heated at 60 °C for 1 h, and dewaxed in xylene (Sigma-Aldrich, St. Louis, MO, USA). The tissue sections were rehydrated in graded ethanol from 95% to 75%, and finally in phosphate buffered solution with 0.05% Tween-20 (PBST). The tissue slides were heated in 10 mM citric acid buffer with 0.05% Tween-20 (pH = 6.0) at 121 °C for 3 min using a pressure cooker for antigen retrieval, and the tissue sections were incubated with peroxidase blocking reagent [RTU, EnVisionTM +Dual Link System-HRP (DAB+) kit, Code K4065, Dako, CA, USA] for 5 min, then blocked with goat serum for 30 min. After blocking, the tissue sections were incubated with antibodies against cyclin D1 (cat. no. GTX108624), PD-1 (cat. no. GTX128435, GeneTex, Irvine, CA, USA), 18hosphor-AKT (cat. no. 4060, Cell Signaling Technology, Beverly, MA, USA), and VEGF (ABS82, Millipore, Bedford, MA, USA) at 4 °C overnight, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. After being rinsed with PBST, the sections were developed in 3′,3′-diaminobenzidine (DAB) substrate kit (Abcam, ab64238), and counterstained with Mayer’s hematoxylin (ScyTek Laboratories, UT, USA). All sections were scanned by an Aperio digital pathology slide scanner (Leica Biosystems, Buffalo Grove, IL, USA).
8
Immunohistochemical Detection of T and B Lymphocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Dako Envision® + Dual Link System-HRP (DAB+) kit (Dako K4965, USA) was used, with a slight modification of the protocol, to detect apoptotic cells in the spleen. Tissue sections, prepared as above, were deparaffinized, rehydrated with ascending concentrations of ethanol (100, 90, and 70%), and washed in distilled water. Wax-enclosed sections were then flooded with dual endogenous enzyme (Dako K4065, USA) as a blocking agent and incubated for 10 min. Sections were then washed with citrate buffer solution (10 mM, pH 6.0) (Sigma, USA), immersed in tris-buffered saline with Tween-20 (TBST) for 3 min, and then incubated with CD3 primary antibody (T-lymphocyte marker) (Abcam ab5690, UK) or CD19 primary antibody (B-lymphocyte marker) (Bioss bs0079R, USA) at 4°C overnight. After washing with TBST, sections were incubated for 45 min with labeled polymer-HRP reagent (Dako K4065, USA) and then washed again with TBST. DAB + substrate-chromogen solution (Dako K4065, USA) was then applied for 3 min. Finally, sections were counterstained with Myer's hematoxylin, mounted in DPX medium, and observed under a light microscope at 40x magnification.
9
Immunohistochemical Profiling of ER Stress Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sections were immunostained with an anti-DDIT3 (9C8) antibody (1:200, Abcam, Tokyo, Japan), anti-IRE1 (phospho S724) antibody (1:500, Abcam), anti-PERK (phospho T980 or phospho T982) antibody (1:500, Abcam), or anti-TGF-β1 antibody (1:1000, Abcam) using the EnVision + Dual Link System/HRP (DAB) Kit (Dako, Tokyo, Japan). Isotype-specific IgG served as the negative control. Antigen retrieval was performed using target retrieval solution (Dako). Immunohistochemistry was performed three independent times using identical samples.
10
Immunohistochemical Analysis of Mouse Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue sections from the indicated mouse models were fixed in 10% buffered formalin and embedded in paraffin. Tissue sections were routinely stained with haematoxylin and eosin. For immunohistochemistry staining, tissue slides were deparaffinized in xylene and rehydrated in alcohol. Endogenous peroxidase was blocked with 3% hydrogen peroxide. Antigen retrieval was achieved using a microwave and 10-mM citric sodium buffer (pH 6.0). Sections were then incubated overnight at 4 °C with the primary antibody. Antibody binding was detected with Envision Dual Link System-HRP DAB kit (K4065, Dako). Sections were then counterstained with haematoxylin. For negative control, the primary antibody was replaced with the buffer. The mitotic index was quantified by viewing and photographing 10 random high-power field of each tissue section on a Nikon microscope, using a 40 × objective. For evaluation and scoring of immunohistochemical data, we randomly selected 10 fields within the tumour area under high-power magnification (× 400) for evaluation. The investigators conducted blind counting for each quantification-related study.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!