The use of the patient samples was approved by Human Ethical Committee of Xiangya Hospital, Central South University. For detection of LMP1 and TAZ, consecutive slide-mounted NPC and gastric cancer sections were first treated with proteinase K at room temperature for 15 min. Endogenous peroxidase activity was inhibited by incubating with 3% H2O2 (DAKO, Carpinteria, CA, USA). Nonspecific binding was blocked with Antibody Diluent and Background Reducing Component (DAKO, Carpinteria, CA, USA). Sections were then incubated with anti-TAZ (CST, Danvers, MA, USA; 1:50 dilution) and anti-LMP1 (DAKO, Carpinteria, CA, USA; 1:100 dilution) antibodies at room temperature for 1 h. After a washing step, a HRP-conjugated secondary antibody was added and sections were incubated at room temperature for 20 min. Tissue sections were then treated with DAB reagent (DAKO, Carpinteria, CA, USA); 3, 3′-Diaminobenzidine tetrahydrochloride was used as a chromogen. All images were acquired on an Olympus BX51 microscope (Leica, Germany).
Dab reagent
Manufactured by Agilent Technologies
Sourced in United States, Denmark
The DAB reagent is a laboratory product used in various immunochemical and histochemical applications. It serves as a chromogenic substrate for detection and visualization of target molecules in biological samples. The core function of the DAB reagent is to catalyze a color-producing reaction, enabling the identification and localization of specific proteins or other analytes of interest.
Automatically generated - may contain errors
Lab products found in correlation
Real envision detection system, by Agilent Technologies (2 mentions)
Bx51 microscope, by Leica (1 mentions)
Anti lmp1, by Agilent Technologies (1 mentions)
Antibody diluent and background reducing component, by Agilent Technologies (1 mentions)
Nanozoomer slide scanner, by Hamamatsu Photonics (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Biotinylated anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Cre recombinase, by Jackson ImmunoResearch (1 mentions)
Cre recombinase, by Fortrea (1 mentions)
Blocking serum, by Santa Cruz Biotechnology (1 mentions)
Envision reagent, by Agilent Technologies (1 mentions)
Ix 70, by Olympus (1 mentions)
Anti ov6, by R&D Systems (1 mentions)
Anti mdm2, by Santa Cruz Biotechnology (1 mentions)
Anti cxcr4, by Abcam (1 mentions)
Altra 20 digital camera, by Olympus (1 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Merck Group (1 mentions)
Universal blocking reagent, by BioGenex (1 mentions)
M0851, by Agilent Technologies (1 mentions)
Axiovision software, by Zeiss (1 mentions)
28 protocols using dab reagent
1
Immunohistochemical Detection of LMP1 and TAZ
2
Immunohistochemical Assessment of Tumor Markers
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PECAM-1(CD31) expression in formalin-fixed paraffin-embedded tumor tissues was assessed as previously described [9 (link)]. Color development was carried out using 3,3′-diaminobenzidine tetrahydrochloride (DAB) reagent (Dako). For Ki67 imaging bright field scanning was performed using a NanoZoomer slide scanner (Hamamatsu). Images were processed using ImageJ (National Institutes of Health) or Imagescope (Aperio) software in a blinded manner as previously described [50 (link), 51 (link)].
3
Immunohistochemical Analysis of EGFL7 and LANA
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Sections of KS biopsy specimen were obtained from the AIDS and Cancer Specimen Resource (ACSR). Tissue sections were subjected to deparaffinization with Histochoice clearing reagent and hydrated with water. The slides were treated with 10mM citrate buffer, pH 3.0, for 30min at 37°C for antigen retrieval then blocked with blocking solution (2% Normal Goat Serum and 0.2% Triton X-100 in PBS). Slides were incubated with primary antibodies, anti-EGFL7 (Abcam Inc.) and anti-LANA (generated at Genscript) separately at 37°C for 1h in a staining chamber followed by treating with HRP-conjugated respective secondary antibodies at 37°C for 1h and developing using DAB reagent (DAKO). The slides were counter-stained with hematoxylin and examined under microscope (Nikon, Inc.) and photographed.
4
Immunostaining of Paraffin Sections
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Sections of paraffin-embedded samples were immunostained as described previously [6 (link)]. Antigen retrieval was performed in 10 mM sodium citrate buffer. Sections were blocked in a solution of Power Block Universal Blocking Agent (Biogenex , Fremont, CA, USA) and incubated with primary antibodies: PyV mT (a generous gift from Dr. S. Dilworth, Ab762, 1:100) or Cre recombinase (Covance, Denver, PA, USA; PRB 106C, 1:600). In the case of PyV mT, the sections were incubated with anti-mouse secondary antibody conjugated to HRP (Dako, Burlington, ON, Canada; #K4006). For Cre recombinase, the sections were incubated first with biotinylated anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories) and then with avidin conjugated to HRP (Vector Labs, Burlington, ON, Canada; Vectastain Elite ABC kit #PK-6100). Development was carried out by exposing with DAB reagent (Dako #K3467). Slides were counterstained with 20% haematoxylin before mounting.
5
Immunohistochemical Analysis of FGFR1 Expression
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IHC for FGFR1 was performed using FGFR1 antibody (Cat, #BA0485, Boster Biological Technology Co. Ltd). Briefly, after deparaffinization and hydration of the sections, the slides were treated with endogenous peroxidase in 0.3% H2O2 for 30 min, followed by blocking for 2h at room temperature with 1.5% blocking serum (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in phosphate-buffered saline (PBS). Subsequently, the slides were probed overnight with FGFR1 antibody (1:100) at room temperature in a moist chamber. Then, the specimens were washed three times in PBS and treated with Envision reagent (Dako), followed by color development using DAB reagent (Dako). Finally, the slides were counterstained with hematoxylin, dehydrated with ethanol, cleaned with xylene, and mounted. As a negative control, duplicate sections were immunostained without exposure to primary antibodies. To quantitate the FGFR1 protein expression, the mean percentage of positive tumor cells was determined in at least five random fields in each section at 400× magnification. The intensity of the FGFR1 immunoreaction was scored as follows: 1+, weak; 2+, moderate; and 3+, intense. The samples with >10% positive tumor cells and the staining at 1+, 2+, and 3+ levels were considered as FGFR1 IHC-positive.
6
Immunohistochemical Analysis of Apoptosis Markers
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The tumors were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 3 µm slices. After the deparaffinizing process, sections were incubated in citrate buffer for 20 min in 70 °C, and retrieval was performed with 2 M HCL for 30 min. Next, 0.3% Triton was used for cell permeability, and blocking was done with 10% goat serum. For the next step, mouse monoclonal anti-Bax or anti-Caspase3 antibody (1:100 dilution, Dako) was added to the slides and incubated overnight at 4 °C followed by secondary antibody incubation in the next day and DAB reagent treatment (1:50 dilution in the buffer, Dako). In the end, counterstaining with hematoxylin was done, and the slides were analyzed by an expert pathologist.
7
Immunohistochemical Profiling of Liver Cancer
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HCC tissue microarray slides were stained with the following primary antibodies at 4 °C overnight: anti-OV6 (R&D Systems), anti-MDM2 (Santa Cruz Biotechnology) and anti-CXCR4 (Abcam). Corresponding secondary antibodies and diaminobenzidine (DAB) reagent (Dako, Carpinteria, CA, USA) were used in the detection procedure. All TMA slides were observed and photographed with an Olympus microscope (IX-70 OLYMPUS, Shinjuku, Tokyo, Japan). The staining levels of OV6, MDM2 and CXCR4 in all clinical samples were examined by two independent observers as described previously.20 (link) The staining intensities were scored as 0 (negative), 1 (weakly positive), 2 (moderately positive) or 3 (strongly positive). Low expression level was defined as score ⩽1, whereas high expression in tumoral tissues was identified as score ⩾2.
8
Immunohistochemistry Protocol for Paraffin Sections
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Paraffin-embedded tissue sections (4 μm) were deparaffinized, subjected to antigen retrieval in hot 10 mM sodium citrate buffer, and then blocked with BSA solution for 1 h at RT. They were incubated with primary antibodies overnight at 4 °C, washed with PBS, incubated with secondary antibodies for 30 min at RT, and developed with DAB reagent (Dako). Sections were counterstained with hematoxylin, dehydrated, and then coverslips were mounted with DPX (Sigma) and slides observed and photographed under the light microscope CX40 Olympus, equipped with the Altra-20 digital camera51 (link).
9
Immunohistochemical Characterization of Tissue Compartments
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Morphological evaluation of the H&E‐stained tissues was supplemented by immunohistochemical staining of the major non‐malignant compartments. Acinar, stromal and immune cells were visualised with antibodies targeting amylase‐A2‐alpha (AMY2A; sheep IgG, abcam #ab18934; Abcam, Camridge, UK), smooth muscle actin alpha (SMACTA2; mIgG2a, DAKO #M 0851; Dako, Hamburg, Germany) and common leucocyte antigen CD45 (mIgG1, DAKO #N1514), respectively. Formalin‐fixed, paraffin‐embedded (FFPE) sections were stained according to a standard protocol.13 In brief, 4 µm‐thick sections were heated to 96°C in citrate buffer (pH 6) for 30 min to retrieve the antigens. They were blocked with methanol containing 3% H2O2 and universal blocking reagent (BioGenex, San Ramon) and exposed to primary antibodies at 4°C overnight. After washing in TBS with 0.05% Tween‐20, slides were exposed for 45 min to anti‐sheep (KPL #5220–0372) or anti‐mouse (DAKO #K400) secondary antibodies labelled with horseradish peroxidase, then incubated with DAB reagent (Dako) for 1 hr and counterstained with hematoxylin. The images were recorded using a light microscope equipped with the AxioVision software (Zeiss, Oberkochen, Germany).
10
Immunohistochemical Analysis of Xenograft Tumor Samples
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