The paraffin specimens were made into 3–5 μm sections. For immunohistochemical staining, paraffin sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. After blocking peroxidase activity with hydrogen peroxide and antigen retrieval by heat induced using citrate buffer (pH 9.0), the sections were incubated at 4°C with primary antibodies against EGFR (1:500, clone 3C6, Ventana Medical Systems Inc., Tucson, AZ, USA), C-erb B2 (1:500, A0485, Dako Cytomation, Glostrup, Denmark), Ki-67 (1:100, MIB-1, Dako Cytomation, Glostrup, Denmark), and p53 (1:100, clone DO-7, Dako Cytomation, Glostrup, Denmark). Immunostaining was conducted using the ImmPRESS (MP7410-50, Vector, Burlingame, CA, USA) system and the diaminobenzidine (DAB) kit (Sk4100, Vector). After the reactions, the sections were counterstained with Meyer’s hematoxylin, dehydrated, cleared, and mounted.
Clone do 7
Manufactured by Agilent Technologies
Sourced in United States, Denmark, Germany
The Clone DO-7 is a laboratory equipment product manufactured by Agilent Technologies. It is a device designed for the measurement and analysis of dissolved oxygen (DO) levels in various applications. The core function of the Clone DO-7 is to provide accurate and reliable DO measurements.
Automatically generated - may contain errors
Lab products found in correlation
Mib 1, by Agilent Technologies (1 mentions)
A0485, by Agilent Technologies (1 mentions)
Clone 3c6, by Roche (1 mentions)
Sk 4100, by Vector Laboratories (1 mentions)
Benchmark xt, by Roche (1 mentions)
Clone 14, by BD (1 mentions)
Clone ov tl 12 30, by Agilent Technologies (1 mentions)
Muc5ac, by Leica (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
Ventana anti her2 neu 4b5, by Roche (1 mentions)
Pr clone pgr 1294, by Agilent Technologies (1 mentions)
Er clone 1d5, by Agilent Technologies (1 mentions)
Clone ep618y, by GeneTex (1 mentions)
Clone 4b11, by Leica (1 mentions)
Clone ep1, by Agilent Technologies (1 mentions)
Tissue preparation system, by Siemens (1 mentions)
Pr clone pgr 636, by Agilent Technologies (1 mentions)
Dako envision flex detection system, by Agilent Technologies (1 mentions)
Dako omnis, by Agilent Technologies (1 mentions)
Clone ve1, by Abcam (1 mentions)
15 protocols using clone do 7
1
Immunohistochemical Characterization of Tumor Markers
2
Immunohistochemical Analysis of p53 and PTEN
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHC for p53 (1:1,000, clone DO-7, DAKO, Santa Clara, CA, USA) and PTEN (1:1,000, clone Y184, Abcam, Cambridge, UK) was performed on 3-μm-thick sections from the TMA block using a Ventana automated immunostainer (BenchMark XT, Ventana Medical Systems, Tucson, AZ, USA), in accordance with the manufacturer's instructions. IHC was interpreted by intensity (negative, weak, and strong) and area (%). The PTEN and p53 expression were classified into three categories: (1) strong staining in ≥10% of the tumor cells was considered as strong positive, (2) samples without any nuclear staining of tumor cells were interpreted as negative, and (3) samples exhibiting weak staining in any area or patchy strong staining in <10% of tumor cells were regarded as weakly positive [28 (link)].
3
Immunohistochemical Profiling of Tumor Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor tissue specimens were formalin-fixed and paraffin-embedded, sectioned (4 μm) and stained with hematoxylin and eosin. Immunohistochemical analyses were performed with a primary polyclonal mouse anti-human antibody directed against trypsin (1:2,000; Qed Bioscience Inc., San Diego, CA, USA), monoclonal mouse anti-human antibodies directed against cytokeratin 7 (1:50; clone OV-TL 12/30; DakoCytomation, Glostrup, Denmark), cytokeratin 18 (1:10; clone DC10; DakoCytomation), synaptophysin (1:2; clone Snp 88; BioGenex, San Ramon, CA, USA), chromogranin A (1:2; clone LK2H10; Linearis Beratungs-GmbH, Wertheim, Germany), Ki-67 (1:100; DakoCytomation), p53 (1:100; clone DO7; DakoCytomation), β-catenin (1:200; clone 14; BD Transduction Laboratories, Lexington, KY, USA) and epidermal growth factor receptor (1:50; clone 31G7; Zymed Laboratories Inc., San Francisco, CA, USA), as well as a polyclonal rabbit anti-human antibody directed against Smad4 (1:50; rabbit polyclonal; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) using the avidin-biotin complex method. If necessary, antigen retrieval in the sections was achieved by microwave pretreatment in citrate buffer (used for trypsin, p53, Smad4 and β-catenin).
4
Immunohistochemical Evaluation of ARID1A and p53
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Loss of ARID1A expression in tumor cell nuclei was used as a surrogate for the presence of ARID1A loss-of-function mutations [9 (link)]. Similarly, p53 immunoreactivity was used as a surrogate for the presence of p53 loss-of-function mutations. The antibodies used in this study were a mouse monoclonal antibody against ARID1A (BAF250a) (Santa Cruz Biotechnology Santa Cruz, CA, USA) and mouse monoclonal antibody against p53 (clone DO-7, DAKO, Carpinteria, CA, USA). Immunohistochemistry for ARID1A and p53 was performed on tissue specimens at a dilution of 1:50 or 1:100, followed by detection using an EnVision+ System with the peroxidase method (DAKO, Carpinteria, CA, USA). The detail protocols for immunostaining and evaluation of ARID1A and P53 have been described in previous reports [9 (link),10 (link)].
5
Immunohistochemical Assessment of Mucin and p53
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mucin and p53 IHC staining was performed by the tyramide signal amplification-avidin-biotin complex method [24 (link)]. We used monoclonal antibodies against MUC5AC (Novocastra, Newcastle-upon-Tyne, UK; diluted 1 : 100) as a marker for gastric faveolar cells, MUC6 (Novocastra; 1 : 100) as a marker for gastric mucous neck cells and pyloric glands, MUC2 (Novocastra; 1 : 100) as a marker for intestinal goblet cells, and p53 (1 : 100, clone DO-7; Dako). The expressions of MUC2, MUC5AC, and MUC6 were regarded as positive when more than 10% of the area was positively stained [17 (link)]. Overexpression of p53 was regarded as positive when more than 10% of tumor cells displayed nuclear immunostaining [25 (link)].
6
Comprehensive IHC Assay for FFPE Specimens
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An immunohistochemistry (IHC) assay of formalin-fixed and paraffin-embedded (FFPE) specimens was performed using Dako’s EnVision System (Glostrup, Denmark). This is a 2-step method in which the application of the primary antibody is followed by a polymeric conjugate consisting of many secondary antibodies bound directly to a dextran backbone, including ER (clone1D5, DAKO), PR (Clone pgR 1294, DAKO), P53 (Clone DO-7, DAKO) and HER2 (VENTANA anti-HER2/neu(4B5), Roche Diagnostics GmbH). Procedures were provided by the manufacturers and completed on a Roche automated IHC instrument (USA). The antibodies and IHC procedures used in this study were part of a standard IHC panel for a pathology laboratory.
7
Immunohistochemical Analysis of Tumor Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumors were fixed in 10% formalin for at least 24hand then embedded in paraffin. Both human and mouse tumor tissues were sectioned at a 5μm thickness and stained with H&E. Immunohistochemistry(IHC) was performed to examine the expression of p53 and DPC4 in the primary human tumors, as previously described [27 (link)], following the protocol of the Department of Diagnostic Pathology at the Asan Medical Center. Briefly, after deparaffinization and antigenic retrieval, the slides were labeled with a monoclonal antibody against p53 (cloneDO-7, 1:3,000; DAKO, Glostrup, Denmark) and DPC4 (clone EP618Y, 1:100; GeneTex, Irvine, CA, USA). Labeling was detected using the avidin-biotin complex staining method. 3, 3′-diaminobenzidine (DAB) was used as the chromogen for p53and 3-amino-9-ethylcarbazole was used for DPC4. A pathologist who was experienced in pancreatic cancer reviewed the slides to compare the tumor architecture and desmoplastic appearance.
8
Molecular Analysis of Tumor DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cases were stained for p53 (clone DO-7, 1:2,000, DAKO), Wilms tumor 1 (WT-1, clone 6F-H1, 1:3,200, Invitrogen), estrogen receptor (ER, Clone EP1, 1:200, DAKO), progesterone receptor (PR, Clone Pgr636, 1:400, DAKO), and CD8 (Clone 4B11, 1:2,000, Novocastra). Procedures and scoring methods are described in the Supplementary Materials and Methods.
Molecular analysis DNA isolation. Tumor DNA was isolated from FFPE-tissue blocks either by using three 0.6-mm tumor cores (n ¼ 16) or by using microdissected tissue from 5 to 10 tissue sections (10 mm; n ¼ 26). DNA isolation was performed fully automated using the Tissue Preparation System (Siemens Healthcare Diagnostics) as described previously (28) . The median tumor cell percentage of the isolated areas was 80% (range, 25%-90%).
Molecular analysis DNA isolation. Tumor DNA was isolated from FFPE-tissue blocks either by using three 0.6-mm tumor cores (n ¼ 16) or by using microdissected tissue from 5 to 10 tissue sections (10 mm; n ¼ 26). DNA isolation was performed fully automated using the Tissue Preparation System (Siemens Healthcare Diagnostics) as described previously (28) . The median tumor cell percentage of the isolated areas was 80% (range, 25%-90%).
9
Pathological Evaluation of Esophageal Adenocarcinoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pathological evaluation was carried out using hematoxylin and eosin-stained sections by two pathologists (KM and MO) in our institution. The presence of carcinoma/dysplasia were evaluated for all patients, and whether the lesion was EAC arising from BE was confirmed for patients with EAC according to the 11th Japanese classification23 (link). All EAC specimens were also immunostained by the anti-p53 antibody. Formalin-fixed paraffin-embedded samples were thin-sliced at 4 µm. The sections were then deparaffinized before the staining procedure. Monoclonal mouse antihuman p53 protein was used as the primary antibody for immunohistochemistry (Clone DO-7, Agilent, CA, USA). The slides were incubated at room temperature for 20 min with the primary antibody. Each EAC case was classified into three subtypes as immunostaining pattern of p53 (overexpression-type mutation, null cell-type mutation, and wild-type patterns).
10
BRAF and p53 IHC Staining in FFPE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sections (4 µm thick) of the original FFPE block used for molecular analysis were subjected to anti-BRAF (mutated V600E) IHC staining (cat. no. ab228461; clone VE1; dilution, 1/100; Abcam) and anti-p53 IHC staining (cat. no. M7001; clone DO-7; dilution, 1/200; Agilent Technologies, Inc.) on a Dako Omnis (Agilent Technologies, Inc.). Heat-induced epitope retrieval was performed using Dako Target Retrieval Solution pH 9 (cat. no. GV804; Agilent Technologies, Inc.) 30 min at 97˚C, followed by primary antibody incubation 20 min at 32˚C and detection with Dako Envision Flex detection system (cat. no. GV800; Agilent Technologies, Inc.) according to the manufacturer's protocol. The sections were counterstained with hematoxylin (cat. no. GC808; Agilent Technologies, Inc.). Control tissues (tonsil for p53 IHC and BRAF V600E mutated tumor for BRAF IHC) and universal negative control antibody (negative control Mouse IgG1; cat. no. X0931; dilution, 1/200; Agilent Technologies, Inc.) were processed in parallel with tissues exposed to the primary as described above.
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!