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Dab substrate system

Manufactured by Agilent Technologies
Sourced in China

The DAB Substrate System is a product offered by Agilent Technologies. It is a laboratory equipment used for the detection and visualization of target proteins in various applications, such as immunohistochemistry and Western blotting. The system provides a stable, soluble substrate that, when exposed to the enzyme-labeled target, produces a brown color reaction for identification and analysis.

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14 protocols using dab substrate system

1

PDGFRα Immunohistochemical Staining

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Sections (5 μm thick) were deparaffinized with xylene before rehydration in an ethanol gradient. Then, endogenous peroxidase activity was quenched by incubating with 3% H2O2 for 10 min. Sections were then blocked with 3% bovine serum albumin and incubated with rabbit anti-PDGFRα at 4 °C overnight. The primary antibodies were subsequently detected by incubation with horseradish peroxidase-conjugated secondary antibodies (Boster, Wuhan, China) at 37 °C for one h. The DAB Substrate System (DAKO) was used to reveal the immunohistochemical staining.
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2

Immunohistochemical Analysis of Ki67 Expression

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Immunohistochemistry for Ki67 (AP10243CM, Gennova) was performed according to the avidin‐biotin‐peroxidase principle (R.T.U. Vectastin Elite ABC kit; Vector Laboratories), as previously described by our group.6 In brief, deparaffinized and rehydrated slides were submitted to heat‐induced antigen retrieval in the microwave for 15 minutes with 10 mmol/L citrate buffer (pH 6.0). After endogenous peroxidase inactivation, incubation with the primary antibody was performed for 2 hours at room temperature. The immune reaction was visualized with 3,3′‐Diamonobenzidine (DAB + Substrate System; Dako). All sections were counterstained with Gill‐2 haematoxylin. For negative controls, primary antibodies were replaced by a universal negative control antibody (N1699, Dako).
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3

Immunohistochemical Analysis of MMPs and Markers

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Five μm-thick paraffin-embedded sections were deparaffinized with xylene (twice for 5 minutes each) before being rehydrated in water using an ethanol gradient. After washing with water, antigen retrieval was performed in a steamer using citrate buffer (pH 6.0, DAKO) for 20 minutes, and the samples were then cooled to room temperature. The sections were then washed with PBST, incubated with 3% H2O2 for 10 minutes and blocked with the avidin/biotin blocker and the serum-free blocking reagent. The sections were subsequently incubated with mouse anti-Mmp2, rabbit anti-Mmp9, rabbit anti-Abcg2 or rabbit anti-Acta2 antibodies overnight at 4 °C. The DAB Substrate System (DAKO) was used to reveal the immunohistochemical staining.
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4

Immunodetection of TUBB3 in Ovarian Cancer

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For each specimen, five to seven paraffin embedded sections were randomly selected. Immunostaining was performed on 3 μm paraffin tissue section mounted poly-l-lysine-coated slides as detailed earlier [4] (link). Briefly, TUBB3 expressing OC cells were identified using the monoclonal anti-human TUBB3 antibody (clone TUJ1; 1:300; Covance). Binding of the monoclonal anti-human TUBB3 antibody was revealed by the EnVision-rabbit+ System-HRP System (Dako, Carpinteria, CA). Diaminobenzidine was the chromogen (DAB Substrate System, DAKO). Negative controls were done by omitting the primary antibody. Microscopic enumeration of TUBB3 expressing tumor cells was done blinded without any prior knowledge of clinical parameters by two authors (GFZ and EM). The proportion of immunostained OC cells was scored at high magnification (40x objective lens) in ten randomly selected tumor areas.
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5

Immunohistochemical Analysis of Tissue Markers

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Four micrometer-thick slides were deparaffinized with xylene before rehydration using an ethanol gradient. Quenching of the endogenous peroxidase activity was achieved by incubating samples with endogenous peroxidase blocker. After blocking with 3% BSA, sections were incubated with mouse anti-α-SMA (Cell Signaling Technology, Boston, MA, USA), mouse anti-TGF-β (R&D, Minneapolis, MN, USA), rabbit anti-VEGFA (Abcam, Cambridge, UK), and mouse anti-FGF basic/FGF2 (Novus, Littleton, CO, USA) overnight at 4 °C. Horseradish peroxidase-conjugated goat anti-mouse/rabbit lgG was used as the secondary antibody. The DAB Substrate System (DAKO) was used to reveal the immunohistochemistry staining.
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6

Immunohistochemical Detection of LHR

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Paraffin-embedded testis tissue sections were soaked in xylene for 10 min two times. Then slides were dehydrated with an alcohol gradient concentration (100%, 95%, 90%, 70%, 50%, and 30% alcohol for 3 min per wash). The slides were incubated with PBS containing 3% H2O2 for 10 min to quench endogenous peroxidase activity. Triton (0.3%) was added to the glass slide for drilling for 10 min. After doing that, the slides were sealed at 37° C for 1 h in PBS containing 3% BSA and incubated with rabbit anti-LHR (Affinity, USA) at 4° C overnight. Whereafter, the slides were incubated in HRP-conjugated secondary antibodies (Boster, Wuhan, China) at 37° C for 1 h. Finally, we used the DAB Substrate System (DAKO) to observe the immunohistochemical staining under a DXM12000F microscope (Nikon, Tokyo, Japan).
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7

Immunohistochemical Staining of α-SMA

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Five-μm-thick paraffin-embedded sections were deparaffinized with xylene (twice for 5 minutes) before being rehydrated in water using an ethanol gradient. After washing with water, antigen retrieval was performed in a steamer using citrate buffer (pH 6.0; DAKO) for 20 minutes, and the samples were then cooled to room temperature. The sections were then washed with TBST buffer and incubated with 3% H2O2 for 10 minutes and blocked with avidin/biotin blocker and serum-free blocking reagent. The sections were subsequently incubated with rabbit anti-α-SMA antibody (Abcam) overnight at 4 °C. The DAB substrate system (DAKO) was used to detect the immunohistochemical staining. 3DHISTECH pannoramic viewer was used for viewing digital slides.
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8

Immunohistochemical Evaluation of Tumor Markers

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MCT1, MCT4, CAIX and Ki67 protein expression for U251 shMCT1 and U251 shCTRL tumours collected from NSG mice were evaluated by immunohistochemistry (IHC). IHC for MCT1 was performed using UltraVision LP detection system HRP Polymer (Thermo Fisher Scientific, Waltham, MA, USA), and for MCT4, CAIX and Ki67 using UltraVision Large Volume Detection System Anti-Polyvalent, HRP (Thermo Fisher Scientific), as previously described [21 (link),23 (link)]. Briefly, deparaffinised and rehydrated slides were submitted to heat-induced antigen retrieval for 20 min at 98 °C with 10 mM citrate buffer (pH 6.0). After endogenous peroxidase inactivation, incubation with the primary antibody was performed overnight for MCT1, and during 2 h for MCT4, CAIX and Ki67, at room temperature. The immune reactions were visualized with 3,3′-diamonobenzidine (DAB + Substrate System; Dako, Denmark) as a chromogen. The slides were counterstained with haematoxylin and mounted with Entellan® (Merck-Millipore, Darmstadt, Germany). For each immunoreaction, a positive control was included.
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9

Immunohistochemical Analysis of V-ATPase and CAXII

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Immunohistochemistry was performed as previously described [43 (link)]. The primary antibodies used were as follows: rabbit anti-V-ATPase (1:100, ab157458 Abcam) and rabbit anti-CAXII (1:200, ab195233, Abcam). IHC was performed with the Ultravision detection system anti-polyvalent, HRP (Lab Vision Corporation). After deparaffinization and rehydration, the slides were heated for 20 min at 98 °C with 10 mM citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidase was inactivated, and the tissues incubated with the primary antibody for 2 h at room temperature. The immune reaction was visualized using the chromogen 3,3′-diaminobenzidine (DAB+ substrate system; Dako). Tissue sections were counterstained with Gill-2 hematoxylin. For negative controls, primary antibodies were replaced by a universal negative control antibody (N1699, Dako). Positive controls were normal colon tissue for CAXII and breast cancer tissue for V-ATPase. Immunoreaction was evaluated using a semiquantitative score system, considering the extension and intensity of staining, as previously described [43 (link)]. Some of the cases could not be evaluated for immunoreaction, due to missing TMA samples or to insufficient representation of the tumor.
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10

Immunohistochemical Analysis of α-SMA and TUNEL Assay

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Five μm-thick slides were deparaffinized with xylene before rehydration using an ethanol gradient. Next, quenching of endogenous peroxidase activity was achieved by incubation with 3% H2O2. After blocking with 3% BSA, the sections were incubated with mouse anti-α-SMA overnight at 4 °C. The secondary antibodies incubated were horseradish peroxidase-conjugated goat anti-mouse IgG (Boster). The DAB Substrate System (DAKO) was used to reveal the immunohistochemical staining.
TUNEL assay as described by the manufacturer (Roche Applied Science, Mannheim, Germany), and the nuclei were stained with 4′ 6-diamidino-2-phenylindole (DAPI) (Sigma). The images were captured using a laser scanning confocal fluorescence microscope (Olympus).
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