After moist heat–induced antigen retrieval with EnVision Flex Target Retrieval Solutions High pH (DakoCytomation), 2.5-μm kidney sections were incubated with antibody to NLRP2 (sc-166584 Santa Cruz Biotechnologies) overnight at 4°C. After washing, sections were incubated with appropriate horseradish peroxidase–conjugated secondary antibodies. Chromogen detection was carried out with the DAB chromogen kit (DakoCytomation). Nuclei were counterstained with hematoxylin, followed by dehydration and mounting.
Dab chromogen kit
Manufactured by Agilent Technologies
Sourced in Denmark, France, Japan
The DAB chromogen kit is a laboratory reagent used for the visualization of specific targets in immunohistochemistry (IHC) and other molecular biology applications. The kit contains a 3,3'-Diaminobenzidine (DAB) solution that reacts with a peroxidase-based detection system to produce a brown colored precipitate, allowing for the localization and identification of the target molecule of interest.
Automatically generated - may contain errors
Lab products found in correlation
Envision system hrp labeled polymer anti rabbit, by Agilent Technologies (4 mentions)
S2003, by Agilent Technologies (4 mentions)
S3022, by Agilent Technologies (3 mentions)
X0909, by Agilent Technologies (2 mentions)
M0737, by Agilent Technologies (2 mentions)
Dako antibody diluent, by Agilent Technologies (1 mentions)
Anti slc12a1, by Abcam (1 mentions)
Ab16491, by Abcam (1 mentions)
Nb100 402, by Novus Biologicals (1 mentions)
Clone fl d6, by Merck Group (1 mentions)
Ab124964, by Abcam (1 mentions)
Positive pixels algorithm, by Indica Labs (1 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Dm4b microscope system, by Leica camera (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Max po, by Nichirei Biosciences (1 mentions)
11 protocols using dab chromogen kit
1
Kidney NLRP2 Immunohistochemistry
2
Immunohistochemical Analysis of Tissue Samples
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IHC was performed on four micron-thick, FFPE sections. FFPE sections were deparaffinized using xylene and rehydrated in graded ethanol. Sections were heated with a pressure cooker to 125 °C for 30 s and 90 °C for 10 s in citrate buffer (pH 6.0) for antigen retrieval. After quenching of endogenous peroxides with 3% H2O2 in methanol, all sections were blocked with 3% BSA for 60 min at room temperature. Sections were then incubated with anti-Ki67 (ab16667, 1:200), anti-pS240/244-S6 (CST#5364, 1:500) antibody overnight at 4 °C. Following primary antibody incubation, sections were incubated with monoclonal mouse anti-rabbit immunoglobulins for 60 min at room temperature. Afterwards, sections were developed using the DAB chromogen kit (Dako #K3468) and lightly counterstained with hematoxylin. The score of the IHC signals was judged by two independent pathologists blindly.
3
Immunohistochemical Quantification of Thick Ascending Limb
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A formalin-fixed and paraffin-embedded 3-μm-thick kidney specimens were placed on ultra plus glass slides. After rehydration, the target antigen epitope heat retrieval was performed on the sections at pH 6 and 95°C for 20 minutes. After blocking endogenous peroxidase, the antigen was detected with the rabbit anti-SLC12A1 (2.5 μg/mL; Abcam, Cambridge, United Kingdom, Cat. #ab191315, dilution 1:100) at 4°C overnight. The immunoreactivity was detected with a secondary antibody (Flex+Mouse Linker) for 30 minutes at room temperature. Both the first and the second antibodies were diluted with the DAKO antibody diluent containing background-reducing components (DAKO, Carpinteria, CA, USA). A DAB chromogen kit (DAKO, Glostrup, Denmark) was used for visualization of immunoreactivity. The sections were counterstained with Mayer hematoxylin, cleared, mounted, and studied under the microscope. The negative control was performed without applying the primary antibody in 1 slide in each of the series of stained slides. Four high-power images of the outer medulla were obtained to measure the height of epithelial lining cells (n = 50 per kidney) of a thick ascending limb (TAL) of the loop of Henle.
4
SPKH1 Expression in Primary and Metastatic ccRCC
5
Immunohistochemical Analysis of Akt and S6 Phosphorylation in Melanoma
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Tissue microarray (ME1004f) containing 18 nevi, 33 metastatic melanomas and 49 malignant melanomas were obtained from Biomax.
Immunohistochemistry was performed on four micron-thick, FFPE sections. FFPE sections were deparaffinized using xylene and rehydrated in graded ethanol. Sections were heated with a pressure cooker to 125°C for 30 seconds and 90°C for 10 seconds in citrate buffer (pH 6.0) for antigen retrieval. All sections were incubated with peroxidase (Dako #S2003) and protein blocking reagents (Dako #X0909) for 5 minutes each. Sections were then incubated with anti-pS473-Akt (CST #4060, 1:50), anti-K140-me3-Akt (1:200), anti-pS420/424-S6 (CST #5364, 1:1500) and anti-SETDB1 (CST #93212, 1:100) antibody diluted in Dako diluent with background reducing components (Dako #S3022) for 1 hr at room temperature. Following primary antibody incubation, sections were incubated with monoclonal mouse anti-rabbit immunoglobulins (Dako #M0737) for 30 minutes at room temperature. Afterwards, sections were incubated with Envision+ System-HRP Labeled Polymer Anti-Rabbit (Dako #K4003) for 30 minutes. All sections were developed using the DAB chromogen kit (Dako #K3468) and lightly counterstained with hematoxylin. The score of the IHC signals was judged by two independent pathologists blindly.
Immunohistochemistry was performed on four micron-thick, FFPE sections. FFPE sections were deparaffinized using xylene and rehydrated in graded ethanol. Sections were heated with a pressure cooker to 125°C for 30 seconds and 90°C for 10 seconds in citrate buffer (pH 6.0) for antigen retrieval. All sections were incubated with peroxidase (Dako #S2003) and protein blocking reagents (Dako #X0909) for 5 minutes each. Sections were then incubated with anti-pS473-Akt (CST #4060, 1:50), anti-K140-me3-Akt (1:200), anti-pS420/424-S6 (CST #5364, 1:1500) and anti-SETDB1 (CST #93212, 1:100) antibody diluted in Dako diluent with background reducing components (Dako #S3022) for 1 hr at room temperature. Following primary antibody incubation, sections were incubated with monoclonal mouse anti-rabbit immunoglobulins (Dako #M0737) for 30 minutes at room temperature. Afterwards, sections were incubated with Envision+ System-HRP Labeled Polymer Anti-Rabbit (Dako #K4003) for 30 minutes. All sections were developed using the DAB chromogen kit (Dako #K3468) and lightly counterstained with hematoxylin. The score of the IHC signals was judged by two independent pathologists blindly.
6
Immunohistochemical Analysis of CTR1 and Nedd4l in Breast Cancer
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Tissue array (BC081120d) containing 10 cases of adjacent normal breast tissues, 100 cases of invasive ductal carcinoma was obtained from Biomax (including ER+ or PR+, 63; HER2+, 32; TNBC, 21). Immunohistochemistry was performed on four micron‐thick, formalin‐fixed paraffin‐embedded (FFPE) sections using an anti‐CTR1 polyclonal antibody (Novus NB100‐402, 1:200) and anti‐Nedd4l polyclonal antibody (Sigma, HPA024618, 1:200). FFPE sections were deparaffinized using xylene and rehydrated in graded ethanol. Sections were heated with a pressure cooker to 125 °C for 30 s and 90 °C for 10 s in citrate buffer (pH 6.0) for antigen retrieval. All sections were incubated with peroxidase (Dako #S2003) and protein blocking reagents (Dako #X0909) for 5 min each. Sections were then incubated with anti‐CTR1 antibody and anti‐Nedd4l antibody diluted in Dako diluent with background reducing components (Dako #S3022) for 1 h at room temperature. Following primary antibody incubation, sections were incubated with monoclonal mouse anti‐rabbit immunoglobulins (Dako #M0737) for 30 min at room temperature. Afterward, sections were incubated with Envision+ System‐HRP Labeled Polymer Anti‐Rabbit (Dako #K4003) for 30 min. All sections were developed using the DAB chromogen kit (Dako #K3468) and lightly counterstained with hematoxylin.
7
Detecting Human Glioblastoma Cells in Mouse Brain
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To detect human glioblastoma cells in mouse brain, we used a 1:100 dilution of the mouse anti-vimentin antibody clone V9 (Dako, M0725), which has no cross-reactivity with mouse vimentin (Valadez et al., 2014 ). We also determined the levels of immune cells positive for CD3-Ɛ using a 1:10 dilution of Armenian Hamster anti-CD3-Ɛ antibody-FITC followed by a 1:100 dilution of the secondary mouse anti-FITC antibody (clone FL-D6, Cat# A1812, Sigma). The HiDefTM HRP-polymer system was used for detection (Cell Marque, Rocklin, CA). Endogenous peroxidase activity was eliminated with treatment under mild conditions as follows: 1.8% H2O2 for 5 min, 1% periodate for 5 min, and 0.02% NaBH4 for 2 min (Polak and Van Noorden, 2003 ). Signal detection was based on the Dako DAB chromogen kit according to the manufacturer's guidelines. We found that hot citric acid and slow cooling of the slide/citrate solution to room temperature (> 20 min) was the best method to retrieve the epitope, but lost epitope if we cooled quickly. The complete methodology can be found in Supplemental Experimental Procedure 1.
8
Liver Fibrosis Quantification Protocol
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Tissues were drop-fixed in 4% paraformaldehyde (Sigma-Aldrich, Saint Quentin Fallavier, France) immediately after collection and put through an automated carousel processor for dehydration, clearing and paraffin embedding (Leica, Nanterre, France). Liver sections (6 μm) were mounted on slides (DPX polymerizing mounting medium Sigma-Aldrich, Saint Quentin Fallavier, France) and stained with H&E and BODIPY. Fibrogenesis was assessed by red picrosirius staining (Red 80, Sigma-Aldrich, Saint Quentin Fallavier, France).
For immunohistochemistry analysis, liver sections were quenched with 3% H2O2, washed with TBS + 0.1% (v/v) Tween-20, blocked with TBS + 3% (w/v) BSA and incubated with diluted primary antibody αSMA (ab124964, Abcam, Paris, France) and then with HRP-conjugated secondary antibody (Bio-Rad, Marnes-la-Coquette, France). Chromogenic detection was carried out using DAB chromogen kit (Dako, Saint Aubin, France). Nuclei were counterstained with hematoxylin. Quantitative expression of all immunostainings was performed using positive pixels algorithm (Indica Labs, Corrales, NM). Results are expressed as percentage of positive pixels. The quantification method is an automated observer-independent process based on section scanning and application of publicly available algorithms. All images were acquired on an Axiovert 200 M microscope (Zeiss, Marly-le-Roi, France).
For immunohistochemistry analysis, liver sections were quenched with 3% H2O2, washed with TBS + 0.1% (v/v) Tween-20, blocked with TBS + 3% (w/v) BSA and incubated with diluted primary antibody αSMA (ab124964, Abcam, Paris, France) and then with HRP-conjugated secondary antibody (Bio-Rad, Marnes-la-Coquette, France). Chromogenic detection was carried out using DAB chromogen kit (Dako, Saint Aubin, France). Nuclei were counterstained with hematoxylin. Quantitative expression of all immunostainings was performed using positive pixels algorithm (Indica Labs, Corrales, NM). Results are expressed as percentage of positive pixels. The quantification method is an automated observer-independent process based on section scanning and application of publicly available algorithms. All images were acquired on an Axiovert 200 M microscope (Zeiss, Marly-le-Roi, France).
9
Immunostaining of AT8 Antigen
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For AT8 immunostaining, antigen retrieval was performed by microwave heating of the sections in sodium citrate buffer (10 mM trisodium citrate, 0.5% Tween-20 in H2O, pH 6.0) for 10 min, followed by incubation with 3% H2O2 for 5 min to inhibit endogenous peroxidase activity. The sections were blocked with 2% goat serum in 1 × TBS with 0.25% Triton-X100 (TBS-X) for 30 min, followed by incubation with a biotinylated primary antibody in blocking buffer overnight at 4 °C. The sections were then labelled with a Dako HRP-linked goat anti-mouse IgG antibody for 1h at room temperature and developed with a Dako DAB chromogen kit. The staining images were captured using Leica DM4B microscope system.
10
Immunohistochemical Analysis of Akt and S6 Phosphorylation in Melanoma
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