Formalin-fixed tumors retrieved from the mice were embedded in wax and sections (5 μm) were prepared using standard methods and stained with eosin and hematoxylin. Sections were processed and stained individually for hematoxylin and eosin staining, ER (1:1, Roche), PR (1:1, Roche), HER2 (1:1, Roche), vimentin (1:400, Leica), E-cadherin (1:50, Dako), CK5/6 (1:50, Dako), GATA3 (1:100, Cell Marque), CK20 (1:5000, Biocare), and GCDFP (1:1000, Cell Marque) before observation by microscopy.
Ck5 6
Manufactured by Agilent Technologies
Sourced in Germany, Denmark
The CK5/6 is a laboratory instrument designed for the analysis and identification of specific proteins in biological samples. It utilizes immunohistochemical techniques to detect and visualize the expression of cytokeratins 5 and 6 in tissue sections or cell preparations. The CK5/6 provides researchers with a tool for studying cell differentiation and lineage in various applications, such as cancer research and tissue characterization.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Agilent Technologies (2 mentions)
Ttf 1, by Agilent Technologies (2 mentions)
Gata3, by Cell Marque (1 mentions)
Dako envision detection kit, by Agilent Technologies (1 mentions)
Ventana benchmark system, by Roche (1 mentions)
Her2 neu, by Agilent Technologies (1 mentions)
Ki 67 mib 1, by Agilent Technologies (1 mentions)
Dako envision visualization system, by Agilent Technologies (1 mentions)
Ttf 1, by Leica (1 mentions)
Bond 3, by Leica (1 mentions)
Horseradish peroxidase, by Agilent Technologies (1 mentions)
Gt vision 3 immunohistochemical assay kit, by Gene Tech (1 mentions)
Axiocam 506 color, by Zeiss (1 mentions)
Zen 2.6 blue edition, by Zeiss (1 mentions)
Ae1 3, by Agilent Technologies (1 mentions)
Dako omnis immunostainer, by Agilent Technologies (1 mentions)
Bond autostainer, by Leica (1 mentions)
Benchmark xt, by Roche (1 mentions)
Alk d5f3 antibody, by Roche (1 mentions)
11 protocols using ck5 6
1
Immunohistochemical Analysis of Tumor Markers
2
Immunohistochemical Staining Protocol
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Immunohistochemical staining was carried out using the DAKO Envision detection kit (Dako, Carpinteria, CA, USA). In brief, paraffin-embedded tissue blocks were sectioned (4 μm-thick), dried, deparaffinized, and rehydrated. Antigen retrieval was performed in a microwave oven for 15 min in 10 mM citrate buffer (pH 6.0). After cells were embedded in 4% neutral formaldehyde for 2 h, PBS with 0.5% Tween-20 was added for 30 min at room temperature. For all samples, endogenous peroxidase activity was blocked with a 3% H2O2-methanol solution. The slides were blocked with 10% normal goat serum for 10 min and incubated with an appropriately diluted primary antibody overnight at 4°C. The slides were then probed with an HRP-labeled polymer conjugated to an appropriate secondary antibody for 30 min. The antibodies against estrogen receptor (ER), progesterone receptor (PR), HER-2, Ki67, cytokeratin 5/6 (CK5/6) and epidermal growth factor receptor (EGFR) were purchased from Dako (Carpinteria, CA, USA) and the GCS antibody was a gift from Dr. D. Marks (Mayo Clinic Center).
3
Comprehensive Immunohistochemical Profiling
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Immunohistochemistry was performed on 3 μm sections derived from formalinfixed paraffin-embedded (FFPE) samples. Samples were immunhistochemically stained by using the Ventana Benchmark system (Roche, Basel, Switzerland) according to the manufacterer´s protocol. Antibodies against cytokeratin 5/6 (CK5/6; 1:50, Dako; Hamburg, Germany), cytokertain 7 (CK7; 1:150, ZytoVision, Bremerhaven, Germany), cytokeratin 14 (CK14; 1:300, DC Systems, Hamburg, Germany), p53 (1:200; Dako, Hamburg, Germany), S100 (1:6000, Dako, Hamburg, Germany), estrogen receptor and progesteron receptor (each 1:20, DC Systems, Hamburg, Germany), Her2/neu (1:300, Dako, Hamburg, Germany), mammaglobin (1:100, Menarini Diagnsotics, Berlin, Germany), muc 4 (1:300, Santa Cruz, Heidelberg, Germany), prostate-specific antigen (PSA; 1:3000, Dako, Hamburg, Germany), androgen receptor (AR; 1:1; CellMarque, Darmstadt, Germany) and Ki67/MIB-1 (1:50, Dako, Hamburg, Germany) were applied.
4
Immunohistochemical Analysis of Lung Adenocarcinoma
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Hematoxylin and eosin (H&E)-stained sections of the tumor were blindly reviewed by three experienced pulmonary pathologists. Immunohistochemical (IHC) staining was performed to exclude mixed and inconspicuous ADC components. The lung tissue sections were deparaffinized three times with xylene and dehydrated through a graded series of ethanol. Endogenous peroxidase activity was quenched with 3% H2O2 in water for 10 min. Antigen retrieval was performed by heating the slides in 0.1 M sodium citrate (pH 6.0) for 10 min. The sections were then incubated with primary antibodies for 30 min at room temperature. Sections incubated with antibody diluents were used as negative controls. The sections were developed using the Dako EnVision™ visualization system (Dako Cytomation, CA, USA), and the following antibodies were used for IHC staining: ΔNP63 (p40; Calbiochem, Darmstadt, Germany) and cytokeratin 5/6 (CK5/6; Dako).
5
Immunohistochemical Analysis of NEDD9 Expression
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Immunohistochemistry was performed on 4 µm thick sections by standard protocols with primary antibodies including TTF-1 (Novocastra, Great Britain), CK7 (Dako, Denmark), napsin A (Novocastra, Great Britain), CK5/6 (Dako, Denmark) and p63 (Novocastra, Great Britain).
The NEDD9 monoclonal antibody (Abcam, San Francisco, CA, USA) was used in 1:400 dilution with EnVision kit for visualization. Normal bronchial epithelium present in tumor surroundings was used as negative control. The ‘hot spot’ was determined by inspection on whole specimen at x40 magnification by two investigators blinded for the clinicopathologic data. The immunohistochemical reaction was examined at x400 magnification.
The percentage of NEDD9 positive tumor cells was assessed semiquantitatively, as follows: 0-25% of positive cells scored 0 or negative; 26%-50% as 1 (weakly positive); 51%-75% as 2 (mildly positive); and more than 75% of positive tumor cells as 3 (strongly positive). Positivity was evaluated separately for nuclei and cytoplasm. The intensity of staining was also scored in the range of 0-3 (negative, weakly stained, mildly or strongly stained) (Fig. 1 ).
The NEDD9 monoclonal antibody (Abcam, San Francisco, CA, USA) was used in 1:400 dilution with EnVision kit for visualization. Normal bronchial epithelium present in tumor surroundings was used as negative control. The ‘hot spot’ was determined by inspection on whole specimen at x40 magnification by two investigators blinded for the clinicopathologic data. The immunohistochemical reaction was examined at x400 magnification.
The percentage of NEDD9 positive tumor cells was assessed semiquantitatively, as follows: 0-25% of positive cells scored 0 or negative; 26%-50% as 1 (weakly positive); 51%-75% as 2 (mildly positive); and more than 75% of positive tumor cells as 3 (strongly positive). Positivity was evaluated separately for nuclei and cytoplasm. The intensity of staining was also scored in the range of 0-3 (negative, weakly stained, mildly or strongly stained) (
6
Histopathological Diagnosis of ASLC
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All surgical specimens were fixed with 10% formalin and embedded in paraffin. Four to six paraffin embedded samples were selected according to the tumor size and involved range, and stained with H&E. ASLC diagnostic criteria were defined as the presence of components of both AD and SCC, with at least 10% of each component. All the specimens were re-evaluated by two senior pathologists. When H&E staining alone failed to diagnose ASLC, the presence of SCC (CK5/6, p40, and p63) and AD [CK7, Thyroid Transcription Factor-1 (TTF-1), and NapsinA] markers was examined using IHC in the adenosquamous region and metastatic lymph nodes by the Super Vision two-step method. The monoclonal antibodies including CK5/6, p63, CK7, and TTF1 were purchased from Dako, Denmark. Meanwhile, p40 and NapsinA were from Jinqiao Biological Company, China. The process of IHC was carried out according to manufacturer instructions.
7
Immunohistochemical Staining Protocol for Neuroendocrine Markers
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Immunohistochemical stainings were carried out using the BOND-III automated immunohistochemical stainer (Leica Biosystems, Nussloch, Germany). All antibodies used in this study were commercially purchased: choromogranin A (IS50230, dilution 1:1000, Dako, Glostrup, Denmark), synaptophysin (PA0299 Bond Ready-To-Use Primary antibody, Leica Biosysytems, Nussloch, Germany) and CD56 (CD56-1B6-R-7 Bond Ready-To-Use Primary antibody, Leica Biosysytems, Nussloch, Germany), Ki67 (M7240, dilution 1:100, Dako, Glostrup, Denmark), c-kit (A4502, dilution 1:100, Dako, Glostrup, Denmark), p53 (N1581, dilution 1:10, Dako, Glostrup, Denmark), p63 (413751, dilution 1:5, Nichirei, Tokyo, Japan), CK5/6 (M7237, dilution 1:250, Dako, Glostrup, Denmark) and CK20 (413491, dilution 1:1000, Nichirei, Tokyo, Japan). Appropriate positive and negative controls were implemented in each reaction. The tumor samples were regarded as positive when more than 10% of nuclei were stained for p53 as previously reported [12 (link)]. The positivity of c-kit was evaluated by the immunoreactivity of cytoplasm in the tumor cells as previously reported [1 (link)]. Each immunostaining was reviewed and independently scored by two pathologists independently (K. T. and R. K.).
8
Generation and Validation of EN1 Antibodies
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Synthetic peptides corresponding to the EN1 candidate regions 27, 28, and 29 (Supplementary Figure S2) were conjugated to keyhole limpet hemocyanin. The conjugated peptides (AbClon, Inc., Seoul, Korea) were then injected into a rabbit and processed according to the manufacturer's instructions. After heat-induced antigen retrieval, slides displaying TMA or cell-block sections were stained with the generated primary EN1 (#27–29, 1:200), CK5/6 (1:100, Dako), and EGFR (1:30, Novocastra) antibodies and labeled with horseradish peroxidase (Dako). In immunohistochemical analysis, the criteria for cytoplasmic versus nuclear staining intensity were essentially the same. Zero denotes essentially no to very little staining, and was categorized as negative. Weak, moderate, and strong staining were defined as positive.
9
Immunohistochemical Analysis of Breast Tissue
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Paraffin-embedded specimens were cut into 4-μm-thick sections and were mounted on polylysine-coated slides, deparaffinized in xylene, rehydrated in a graded series of alcohol solutions, and stained with H&E. For immunohistochemical analysis, the slides were heated in 10 mM sodium citrate (pH 6.5) with a pressure cooker for 10 min. After being treated with 3% H2O2 for 5 min and blocked with 10% normal goat serum for 30 min, the sections were probed with primary antibodies against ER-α, PR, HER2, E-cadherin, CK5/6, EGFR, P120, or Ki-67 (Dako) for 1 h at room temperature. Detection of the primary antibody and color development were performed using the GT vision III Immunohistochemical Assay Kit (GK500710; Gene Tech) according the manufacturer’s protocol. The sections were counterstained with hematoxylin.
10
Comprehensive Histological Characterization Protocol
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After fixation for at least 24 hr in 10% neutral buffered formalin, the tissue samples were dehydrated in a graded series of ethanol and xylene, mounted in paraffin and cut into 3-μm-thick sections. In addition to hematoxylin and eosin (HE), Elastica-van-Gieson (EvG), Berlin Blue (Fe), periodic acid Schiff stain (PAS), Alcian Blue-periodic acid Schiff stain (abPAS), Giemsa, Gomori Trichrome, and Kongo Red stain were used by following routine protocols. For immunohistochemistry, the following antibodies were used: AE1/3 (Dako/IR053), TTF-1 (Dako/IR056), CK7 (Dako/IR619), CK5/6 (Dako/IR780), p40 (Zytomed/MSK097), Ki67 (Dako/IR626), CD68 (Dako/M0876), CD61 (Dako/M0753), CD31 (Dako/IR610), CD34 (Dako/IR623), ASMA (Dako/IR611), CD3 (Dako/IR503), CD20 (Dako/IR604), MUM1 (Dako/IR644), collagen IV (Dako/M0785), and tenascin (Chemicon/MAB19101). All immunostaining were performed with the Dako Omnis immunostainer (Agilent) by following routine procedures. The sections were examined microscopically (Axio Imager. M2, Carl Zeiss Microscopy GmbH), and representative photographs were obtained (Axiocam 506 color, Carl Zeiss Microscopy GmbH; ZEN 2.6 (blue edition), Carl Zeiss Microscopy GmbH).
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