Sixty-two formalin-fixed paraffin-embedded (FFPE) tissues from bladder cancer patients of various stages and grades were collected from Hospital Kuala Lumpur. The cohort of tissues comprised of 17 Grade 1, 18 Grade 2, and 27 Grade 3 tumours, of which, there were 18 pTa, 19 pT1, 11 pT2, and 14 pT3 and pT4 stages. Tissues were deparaffinized at 36°C for 1 hour. Slides were sequentially rehydrated with 100%, 95%, 80%, and 70% ethanol immersions, followed by antigen retrieval which was done by boiling the slides in sodium citrate using a microwave. The sections were then incubated with a primary polyclonal rabbit antibody (ab85567) against CERS2 (Abcam, Cambridge, United Kingdom) at 4°C overnight and then incubated with Dako DAB chromogen (Dako, Glostrup, Denmark) secondary antibody at room temperature for 10 minutes. A negative control was run in every experiment by replacing the primary antibody with 10% TBS. Sections were finally counterstained using haematoxylin and sequentially dehydrated with 70%, 80%, 95%, and 100% ethanol immersions, mounted with DPX, and then observed under the microscope. Scoring was based on staining intensity.
Dako dab chromogen
Manufactured by Agilent Technologies
Sourced in Denmark
The Dako DAB chromogen is a laboratory reagent used for the visualization of immunohistochemical staining reactions. It provides a brown color when reacted with the enzyme-labeled detection system, allowing for the identification of target antigens in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab85567, by Abcam (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Diluent buffer, by Agilent Technologies (1 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Dako autostainer, by Agilent Technologies (1 mentions)
Dako lsab2 streptavidin hrp, by Agilent Technologies (1 mentions)
Biotinylated goat anti rabbit immunoglobulin g igg, by Vector Laboratories (1 mentions)
Dako lsab2 streptavidin horseradish peroxidase, by Agilent Technologies (1 mentions)
Sigma citrate buffer, by Merck Group (1 mentions)
Vector horseradish peroxidase streptavidin, by Vector Laboratories (1 mentions)
Thermo autostainer 360, by Thermo Fisher Scientific (1 mentions)
Avidin d and biotin solutions, by Vector Laboratories (1 mentions)
Goat anti rabbit igg, by Vector Laboratories (1 mentions)
Ab32678, by Abcam (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Axioskop microscope mrc5, by Zeiss (1 mentions)
8 protocols using dako dab chromogen
1
CERS2 Expression in Bladder Cancer
2
Immunohistochemistry Protein Detection
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Primary antibodies and experimental conditions are listed in table 2 . Immunohistochemistry was performed on 5-µm-thick formalin-fixed paraffin-embedded sections. Endogenous peroxidase activity was blocked with 1% hydrogen peroxide. The sections were incubated with primary antibodies in diluent buffer (Dako, UK Ltd, Ely, UK) followed by 30 minutes of incubation at room temperature with secondary antibodies. Immunocomplexes were visualized with Dako DAB+ Chromogen (Dako, Glostrup, Denmark). Sections were washed with phosphate-buffered saline (Fisher Scientific, Ltd, Leicestershire, UK) between all steps. Negative controls were run simultaneously but without primary antibodies.
3
Immunohistochemical Analysis of Ebola Virus
4
Ebola Virus Necropsy and Immunohistochemistry
5
Immunohistochemical Staining for Nipah Virus
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Tissue sections were deparaffinized and rehydrated through xylene and graded ethanol washes. Slides went through heat antigen retrieval in a steamer at 95°C for 20 minutes in Sigma Citrate Buffer, pH 6.0, 10× (Sigma-Aldrich). To block endogenous peroxidase activity, slides were treated with 3% hydrogen peroxide and rinsed in distilled water. The tissue sections were processed for IHC using the Thermo Autostainer 360 (Thermo Fisher Scientific). Sequential 15-minute incubations with avidin D and biotin solutions (Vector Laboratories, SP-2001) were performed to block endogenous biotin reactivity. Specific anti-NiV immunoreactivity was detected using an anti-NiV m102.4 human monoclonal antibody (53 (link)) at a 1:4,000 dilution for 60 minutes. Secondary antibody was biotinylated goat anti-rabbit IgG (Vector Laboratories, BA-1000) at 1:200 for 30 minutes followed by Vector Horseradish Peroxidase Streptavidin (ready-to-use; Vector Laboratories, SA-5704) for 30 minutes. Slides were developed with Dako DAB chromogen (Dako, K3468) for 5 minutes and counterstained with hematoxylin for 45 seconds.
6
Immunohistochemical Analysis of pCaMKII-T286
7
Immunohistochemical Analysis of Immune Markers
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The tissue was fixed in 10% neutral buffered formalin and embedded in paraffin, after which 4–5μ thick sections were cut and mounted onto Plus slides (Fisherbrand, Pittsburg, PA). These sections were baked for 60 minutes at 60°C, deparaffinized, rehydrated, and submitted to antigen retrieval by using 1 mM EDTA, pH 8.0 for 20 minutes in a steamer, followed by 20 minutes of cooling to room temperature. All staining steps were performed on the Dako Autostainer Plus Agilent Technologies, US Headquarters Santa Clara, CA. Endogenous peroxidase activity was quenched by using 3% hydrogen peroxide for 10 minutes, and the slides were washed with Dako's Wash Buffer. Primary antibodies for MCT (AA1, M7052; Dako North America, Carpenteria, CA) and IL-4, IL-5, IL-13, GM-CSF, and TGF-β (C-19, sc-1292; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), in 1:200 dilution were applied for 60 minutes. By using Dako's Envision + Polymer secondary antibody (30 minutes at room temperature), the Dako DAB+ chromogen (Agilent Technologies) was applied to visualize the staining pattern. The slides were counterstained with Dako's Mayer's hematoxylin (Agilent Technologies), and the coverslip was mounted with Permount (Fisherbrand, Pittsburg, PA). The staining was interpreted as none, 1+ (weak), 2+ (moderate) or 3+ (strong), and the numbers of cells were interpreted as low, moderate, and high.
8
Placental Graft Histological Evaluation
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Morphology of the tissue before and after engraftment was evaluated with different stainings: hematoxylin and eosin (HE), hCG, and anti-murine CD31 (also known as platelet-endothelial cell adhesion molecule-1 (PECAM-1)). Placental grafts were stored for 24h in 4% buffered formaldehyde, washed with phosphate buffered saline (PBS), transferred to ethanol 70% and embedded in paraffin.
Four µm sections were deparaffinated and rehydrated for HE and immunohistochemical expression of hCG and murine CD31. hCG antigen retrieval was performed In Tris-EDTA buffer at pH9, at 80°C for 45 minutes. Sections were rinsed in PBS and incubated at room temperature respectively for 2h and overnight with following primary antibodies hCG (3,55μg/ml, polyclonal, Agilent Technologies, California, USA) and CD31 (15,6μg/ml, monoclonal, BD biosciences, New Jersey, USA). Binding of species-specific biotinylated secondary antibodies was visualized with Dako DAB + Chromogen (K3467, Agilent Technologies, California, USA). Counterstaining was performed with Mayer's hematoxylin before mounting. Negative controls were performed by omitting the primary antibody. Positive staining was identified by the presence of a cytoplasmic trophoblastic or membranous endothelial brown (DAB) reaction product. Images were taken using the Axioskop microscope (MRc5, Zeiss, Jena, Germany) and the ZEN 2.0 software.
Four µm sections were deparaffinated and rehydrated for HE and immunohistochemical expression of hCG and murine CD31. hCG antigen retrieval was performed In Tris-EDTA buffer at pH9, at 80°C for 45 minutes. Sections were rinsed in PBS and incubated at room temperature respectively for 2h and overnight with following primary antibodies hCG (3,55μg/ml, polyclonal, Agilent Technologies, California, USA) and CD31 (15,6μg/ml, monoclonal, BD biosciences, New Jersey, USA). Binding of species-specific biotinylated secondary antibodies was visualized with Dako DAB + Chromogen (K3467, Agilent Technologies, California, USA). Counterstaining was performed with Mayer's hematoxylin before mounting. Negative controls were performed by omitting the primary antibody. Positive staining was identified by the presence of a cytoplasmic trophoblastic or membranous endothelial brown (DAB) reaction product. Images were taken using the Axioskop microscope (MRc5, Zeiss, Jena, Germany) and the ZEN 2.0 software.
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