Clone e6h4

Manufactured by Roche

Sourced in United States, Switzerland

The Clone E6H4 is a laboratory equipment product designed for use in various research and diagnostic applications. It serves as a core functional component without further interpretation or extrapolation on its intended use.

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Lab products found in correlation

Close spelling variants of this product

CINtec® p16INK4a Histology Kit (E6H4 clone) (4 citations) Anti-human p16 monoclonal antibody (clone: E6H4, (2 citations)

17 protocols using clone e6h4

Immunohistochemical Scoring of Cervical Lesions

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All H&E and immunohistochemically stained slides were scored by a gynecopathologist (M.C.G.B.) and a senior resident in pathology (N.B.T.).
The Optiview detection kit with the automated 100 BenchMark ULTRA IHC/ISH system (Roche) was used to perform immunostaining of both p16^INK4a and Ki-67. Mouse monoclonal antibodies against the p16^INK4a antigen (clone E6H4; Roche, Basel, Switzerland) were used for immunostaining of p16^INK4a. Immunostaining of Ki-67 was performed with mouse monoclonal antibodies against the Ki-67 antigen (clone Ki-67; Dako, Glostrup, Denmark).
A negative or patchy staining pattern of p16^INK4a was scored as 0, whereas a diffuse (or block) p16^INK4a staining pattern up to the lower third of the epithelium was scored as 1, extending above the lower third of the epithelium was scored as 2, or extending more than two-thirds of epithelium was scored as 3. When a diffuse staining pattern for p16^INK4a was present, we also scored whether this pattern was completely diffuse or combined with a negative or patchy staining pattern. Ki-67 expression was scored as not increased (score 0), increased in the lower third (score 1), increased in the lower two-thirds (score 2), or increased in more than two-thirds (score 3) of the epithelium.

Thuijs N.B., Schonck W.A., Klaver L.L., Fons G., van Beurden M., Steenbergen R.D, & Bleeker M.C. (2021). Biomarker Expression in Multifocal Vulvar High-Grade Squamous Intraepithelial Lesions. Cancers, 13(22), 5646.

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Western Blot Analysis of p16INK4A

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Whole‐cell protein extracts of HeLa and HeLa16 cells were separated in 10% SDS‐PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). The protein bands were first immunoreacted with our novel p16^INK4A‐specific monoclonal antibodies (1:500, clones 1H1087, 1A72 and 1B517), an immunochemistry‐purposed mouse anti‐human p16^INK4A monoclonal antibody (1:500, clone E6H4; Roche, Basel, Switzerland) or an anti‐human β‐actin antibody (1:500; CW Biotechnology, Beijing, China). The immunoreactions were then detected with a (secondary) HRP‐conjugated rabbit anti‐mouse IgG polyclonal antibody (1:500; Abcam, Cambridge, MA, USA) and visualised with ECL Western Blotting Detection reagents (GE Healthcare).

He Y., Shi J., Zhao H., Wang Y., Zhang C., Han S., He Q., Li X., Li S., Wang W., Yi M., Hu X., Xing Z., Han H., Gao Y., Zhou Q., Lu L., Guo J., Cao H., Lu C., Hou Y., Chen D., Yang F., Lei P., Di W., Qian J., Xia Y., Zhang Y., Deng Y., Zhu J, & Xu C. (2023). p16INK4A flow cytometry of exfoliated cervical cells: Its role in quantitative pathology and clinical diagnosis of squamous intraepithelial lesions. Clinical and Translational Medicine, 13(3), e1209.

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Evaluating p16 Expression in FFPE Samples

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Fixed and paraffin-embedded samples were evaluated by p16^INK4a immunohistochemistry (IHC) using clone E6H4 of CINtec (Roche, Heidelberg, Germany) to determine the p16 status of a tumor specimen. The sample was considered positive for p16 if diffuse nuclear and cytoplasmic staining were present, and > 70% of cells were stained.

Yoo S.H., Ock C.Y., Keam B., Park S.J., Kim T.M., Kim J.H., Jeon Y.K., Chung E.J., Kwon S.K., Hah J.H., Kwon T.K., Jung K.C., Kim D.W., Wu H.G., Sung M.W, & Heo D.S. (2018). Poor prognostic factors in human papillomavirus-positive head and neck cancer: who might not be candidates for de-escalation treatment?. The Korean Journal of Internal Medicine, 34(6), 1313-1323.

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Cervical Lesion Characterization via IHC

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All tissue blocks were cut to provide sections of 3 µm. The first and last sections were used for H&E staining to ensure the presence of the same cervical lesion, and in-between sections were used for immunostaining with mouse monoclonal antibodies against Ki-67 antigen (DAKO, Clone MIB-1) or p16^ink4a antigen (Roche, CINtec, Clone E6H4), using the automated IHC Ventana staining machine (Roche, Basel, Switzerland).

van Zummeren M., Leeman A., Kremer W.W., Bleeker M.C., Jenkins D., van de Sandt M., Heideman D.A., Steenbergen R., Snijders P.J., Quint W.G., Berkhof J, & Meijer C.J. (2018). Three-tiered score for Ki-67 and p16ink4a improves accuracy and reproducibility of grading CIN lesions. Journal of Clinical Pathology, 71(11), 981-988.

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Immunohistochemical Profiling of HPV Lesions

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Serial sections of 3 μm were cut from all tissue blocks. To ensure the presence of the same lesion in all specimens, the first and last sections were stained for H&E (sandwich technique). In between sections were immunostained with mouse monoclonal antibodies (mAb) against Ki-67 antigen (Clone MIB-1, DAKO, Denmark) or p16 ink4a antigen (Clone E6H4 ™ , CINtec ® , Roche, Switzerland) by the automated IHC Ventana staining machine (Ventana Medical Systems, Roche, USA), or with the validated mAb panHPVE4 (further referred to as "E4", mAb FH1.1, produced in the laboratory of J. Doorbar, previously described by van Baars et al., reactive against high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59 , 66, 67, and 70) [2, 12] . For E4 staining, slides were deparaffinized in xylene and rehydrated in a descending alcohol series. FH1.1 primary antibody was applied at a concentration of 1:500 after 30-min microwave pretreatment in Tris/EDTA buffer (10 mM Tris/1 mM EDTA, pH 9.0) and incubated overnight at 4 °C. Application of the primary antibody was followed by incubation with BrightVision plus Poly-HRP anti-mouse IgG (ImmunoLogic, The Netherlands), diaminobenzidine as a chromagen and Hematoxylin nuclear counterstaining.

Zummeren M.V., Kremer W.W., Leeman A., Bleeker M.C.G., Jenkins D., Sandt M.V., Doorbar J., Heideman D.A.M., Steenbergen R.D.M., Snijders P.J.F., Kenter G.G., Quint W.G.V., Berkhof J, & Meijer C.J.L.M. (2018). HPV E4 expression and DNA hypermethylation of CADM1, MAL, and miR124-2 genes in cervical cancer and precursor lesions. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 31(12).

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Immunohistochemical Evaluation of Tumor Markers

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Four-μm-thick sections of FFPE tumors were pretreated and immunostained using the Ventana Benchmark XT system. Primary antibodies for MDM2 (clone IF2, Invitrogen, dilution 1/100), CDK4 (clone DCS-31, Invitrogen, dilution 1/200), and p16 (clone E6H4, Roche, ready-diluted) were used as previously described [5, [19] [20] [21] [22] .
The results were independently evaluated by 3 pathologists (NS, SFKJ and ST) who were blinded to the final diagnosis; discordant cases were reevaluated collegially. For each antibody, only well-defined nuclear reactivity was considered positive, whereas ill-defined, perinuclear and cytoplasmic staining were disregarded.
A tumor was considered positive for MDM2 or CDK4 when at least one tumor cell nucleus was stained per low power field, as previously described [5] . p16 IHC was semiquantitatively

Kammerer-Jacquet S.F., Thierry S., Cabillic F., Lannes M., Burtin F., Henno S., Dugay F., Bouzillé G., Rioux-Leclercq N., Belaud-Rotureau M.A, & Stock N. (2017). Differential diagnosis of atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma: utility of p16 in combination with MDM2 and CDK4 immunohistochemistry. Human pathology, 59.

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Immunohistochemical Staining of p16 and p53

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Sequential 4 µm thick (FFPE) sections were stained for p16 and p53 using Ultraview universal DAB detection Kit (#760-700, Ventana). In brief, following deparaffinization and heat-induced antigen retrieval with CC1 (#950-500, Ventana) for 64 minutes, tissue samples were incubated with either anti-p16 (1.0 µg/ml ready to use, clone E6H4, Ventana) or anti-p53 (2.5 µg/ml ready to use, clone BP53-11, Ventana) for 32 minutes at 37°C. Incubation was followed by a hematoxylin II counter stain for 8 minutes and then a blue coloring reagent for 8 minutes according to the manufacturer’s instructions (Ventana).

Spoor J.K., den Braber M., Dirven C.M., Pennycuick A., Bartkova J., Bartek J., van Dis V., van den Bosch T.P., Leenstra S, & Venkatesan S. (2024). Investigating chromosomal instability in long-term survivors with glioblastoma and grade 4 astrocytoma. Frontiers in Oncology, 13, 1218297.

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Immunohistochemical Staining of p16 and p53

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Immunohistochemical (IHC) staining was performed on 4-μm sections of TMA using the Ventana autostainer and UltraView DAB detection kit (Ventana Medical Systems, Tucson, AZ, USA), according to the manufacturer’s instructions. The antibodies we used were p16INK4a (1:6, clone E6H4, mouse mAb, Ventana Medical Systems) and p53 (1:1,500, clone M7001, mouse mAb, Dako, Glostrup, Denmark). According to the eighth edition of the AJCC cancer staging manual, p16 immunostaining was positive when it showed greater than a +2/+3 intensity in > 75% of tumor cells. Separately, the result of p53 was positive if nuclear staining was present in > 10% of tumor cells.

Lee M., Jo U., Song J.S., Lee Y.S., Woo C.G., Kim D.H., Kim J.Y., Yoon S.O, & Cho K.J. (2023). Clinicopathologic characterization of cervical metastasis from an unknown primary tumor: a multicenter study in Korea. Journal of Pathology and Translational Medicine, 57(3), 166-177.

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HPV Genotyping and p16 Immunohistochemistry

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As described previously^{32 (link),33 (link)}, the DNA-extractions from the samples were analysed for presence of 40 different HPV subtypes using a multiplex Modified General Primers (MGP)-PCR and Luminex. In addition, HPV positive lesions were also subjected to immunostaining for p16. Four µm sections of FFPE tissue were prepared utilising the CINtec p16-kit, Clone E6H4, (Ventana Medical Systems), and the BenchMark Ultra platform (Ventana Medical Systems). Positive p16 expression was defined as at least 70% nuclear and cytoplasmic expression with moderate to strong intensity^{34 (link)}.

Nilsson J.S., Forslund O., Andersson F.C., Lindstedt M, & Greiff L. (2019). Intralesional EBV-DNA load as marker of prognosis for nasopharyngeal cancer. Scientific Reports, 9, 15432.

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Immunohistochemical Analysis of Cell Markers

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Immunohistochemical studies were performed on 4-μm formalin-fixed paraffin-embedded sections with commercially available antibodies targeting the following proteins: Rb (1:100 dilution, clone G3–245; BD Biosciences, San Jose, CA, USA), MDM2 (1:200 dilution, clone IF2; MilliporeSigma, Burlington, MA, USA), p16 (1:4 dilution, clone E6H4; Ventana Medical Systems, Roche, Indianapolis, IL, USA), p53 (prediluted, clone DO-7; Ventana Medical Systems), and CD34 (1:160 dilution, clone MY10; BD Biosciences). Immunostaining for Rb, p16 and CD34 was performed on the Leica Bond (Leica, Buffalo Grove, IL, USA), and immunostaining for p16 and p53 was performed on the Ventana BenchMark ULTRA (Ventana Medical Systems). Appropriate positive and negative controls were used throughout.

Hammer P.M., Kunder C.A., Howitt B.E, & Charville G.W. (2022). Well-differentiated lipomatous neoplasms with p53 alterations: a clinicopathological and molecular study of eight cases with features of atypical pleomorphic lipomatous tumour. Histopathology, 80(4), 656-664.

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