Clone 4b5

Manufactured by Roche

Sourced in United Kingdom, United States

The Clone 4B5 is a laboratory instrument used for cell culture applications. It is designed to provide a controlled and consistent environment for the growth and maintenance of cells. The core function of the Clone 4B5 is to maintain stable temperature, humidity, and gas composition within the incubation chamber to support optimal cell culture conditions.

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Lab products found in correlation

Clone 1e2, by Roche (4 mentions) Clone sp1, by Roche (4 mentions) Mib 1, by Agilent Technologies (3 mentions) Ultraview detection kit, by Roche (3 mentions) Clone 44, by Cell Marque (3 mentions) Clone 138g6, by Cell Signaling Technology (3 mentions) Clone m1, by Roche (3 mentions) Clone mrq28, by Cell Marque (3 mentions) Pgr 636, by Agilent Technologies (2 mentions) Benchmark xt, by Roche (2 mentions) Clone sp1, by Lab Vision (2 mentions) Ep38y, by Abcam (2 mentions) Clone sp44, by Roche (2 mentions) Clone g219 1129, by Roche (2 mentions) Clone 6f11, by Roche (1 mentions) Dako envision dual link system hrp, by Agilent Technologies (1 mentions) Dako envision kit hrp, by Agilent Technologies (1 mentions) Her2 iqfish pharmdx, by Agilent Technologies (1 mentions) Clone 30 9, by Roche (1 mentions) Anti her2 neu, by Roche (1 mentions)

Close spelling variants of this product

Anti-HER2 (clone 4B5, HER2 PATHWAY Clone 4B5 Anti-intracellular domain (ICD) HER2 clone 4B5 HER2 (clone 4B5; Anti-HER2 antibody (clone 4B5, ERBB2 (clone 4B5, Anti-HER-2/neu rabbit monoclonal antibody clone 4B5 Anti-HER2/neu antibody (clone number 4B5,

14 protocols using clone 4b5

Biomarker Concordance in Primary and Metastatic Breast Cancer

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Fixation times ranged from six to 48 hours. Immunohistochemical (IHC)
stains for ER, PR and HER2 were performed in 5 μm-thick whole tissue
sections, or in cell blocks (CBs) fixed in CytoLyt® or formalin. FDA
approved monoclonal antibodies for ER (Ventana; clone 6F11), PR (Ventana; clone
1E2), and HER2 (Ventana; clone 4B5) were utilized for IHC testing, with positive
and negative controls. The hematoxylin and eosin (H&E) and IHC slides
were re-reviewed by 2 pathologists (ME and RDJ); interpretation of ER, PR and
HER2 IHC, and HER2 fluorescence in situ hybridization (FISH) results were done
following the ASCO/CAP guidelines.^{14 (link),15 (link)} ER and PR
discordance between the PBC and the MBC was defined as a switch in the receptor
status from positive to negative, or from negative to positive. HER2 status was
determined following the integrated evaluation of IHC and FISH results. HER2
discordance between the PBC and MBC was defined as a switch from HER2 positive
to negative, or HER2 negative to positive. Cases with HER2 status changes
between the PBC and MBC from positive or negative to equivocal, or vice-versa,
were considered concordant.

Pareja F., Murray M.P., Jean R.D., Konno F., Friedlander M., Lin O, & Edelweiss M. (2017). Cytologic assessment of estrogen receptor, progesterone receptor, and HER2 status in metastatic breast carcinoma. Journal of the American Society of Cytopathology, 6(1), 33-40.

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Immunohistochemical Profiling of Breast Cancer

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The FFPE specimens from the 92 patients were examined. Tissue sections were deparaffinized and hydrated through graded alcohols and xylene. Antigen retrieval was carried out with citrate buffer at pH 6.0 in an autoclave at 121°C for 10 minutes. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 minutes. After rinsing and blocking with 5% normal donkey serum, the sections were incubated overnight at 4°C with primary Ab. The sections were washed and incubated for 2 hours at 4°C with the Dako EnVision + Dual Link System‐HRP (Dako). Diaminobenzidine (Dako EnVision kit/HRP [DAB]) was used for detection of protein. The sections were finally counterstained with hematoxylin. On IHC, ER status and PgR status were assessed semiquantitatively and reported as positive when more than 1% of the nuclei of cancer cells showed staining. Positive HER2 status was determined if strong staining of the complete membrane in more than 10% of tumor cells was observed. Details of Abs are as follows: ER, rabbit monoclonal, clone SP1 (Ventana); PgR, rabbit monoclonal, clone 1E2 (Ventana); and HER2, rabbit monoclonal, clone 4B5 (Ventana). For Ki‐67 staining, mAb (MIB‐1; Dako) (1:400) was used and the cells positive for nuclear Ki‐67 were counted in at least 500 cancer cells in one hotspot on each sample.

Murakami F., Tsuboi Y., Takahashi Y., Horimoto Y., Mogushi K., Ito T., Emi M., Matsubara D., Shibata T., Saito M, & Murakami Y. (2020). Short somatic alterations at the site of copy number variation in breast cancer. Cancer Science, 112(1), 444-453.

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HER2 Status Evaluation in Breast Cancer

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The primary breast cancer tissue sample for each patient was retested for HER2 status to ensure proper classification as HER2-negative. HER2 protein overexpression was evaluated by immunohistochemistry (IHC) using an FDA-approved monoclonal antibody (clone 4B5, Ventana) directed against the internal domain of the c-erbB-2 oncoprotein (HER2). The IHC results were categorized as follows: 0, 1+ = negative result; 2+ = equivocal result; and 3+ = positive result, according to the published American Society of Clinical Oncology (ASCO) guidelines [8 ] (Table 1). Tissues with 2+ staining (equivocal) were assessed for HER2 amplification with fluorescence in situ hybridization (FISH) as per ASCO guidelines [8 ] using an FDA-approved probe set (HER2 IQFISH pharmDx™, Dako), and defined as HER2/CEP17 ratio ≥ 2.0. Carcinoma with 0 or 1+ IHC staining or 2+ IHC staining and concurrent negative HER2 FISH (HER2/CEP17 ration < 2.0) were classified as HER2-negative primary breast malignancies.

Ulaner G.A., Hyman D.M., Lyashchenko S.K., Lewis J.S, & Carrasquillo J.A. (2017). 89Zr-trastuzumab PET/CT for detection of HER2-positive metastases in patients with HER2-negative primary breast cancer. Clinical nuclear medicine, 42(12), 912-917.

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Biomarker Immunohistochemistry in Breast Cancer

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Immunohistochemical staining results for standard biomarkers including ER, PR, HER2, p53, and Ki-67 were searched during the study to identify any data missing from the pre-chemotherapeutic biopsy and post-chemotherapeutic resection specimens. In cases with missing data, immunohistochemical staining on representative tissue sections was carried out in a BenchMark XT autostainer (Ventana Medical Systems, Tucson, AZ) using an UltraView detection kit (Ventana Medical Systems). The following antibodies were used: ER (1:100, clone SP1, Labvision, Fremont, CA), PR (1:70, PgR636, Dako, Carpinteria, CA), HER2 (ready to use, clone 4B5, Ventana Medical Systems), p53 (1:600, D07, Dako), and Ki-67 (1:250, MIB-1, Dako).
For each case, immunohistochemical slides for basic biomarkers were reviewed to acquire information about biomarker expression. ER and PR were regarded as positive if there were at least 1% positive tumor nuclei. ER and PR were also scored using the Allred scoring system [17 (link)]. HER2 expression was scored according to 2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines [18 (link)]. For p53, cases with 10% or more positive staining were grouped as positive. For the Ki-67 proliferation index, cases with 20% or more positive tumor cells were regarded as having high indices.

Ahn S., Kim H.J., Kim M., Chung Y.R., Kang E., Kim E.K., Kim S.H., Kim Y.J., Kim J.H., Kim I.A, & Park S.Y. (2018). Negative Conversion of Progesterone Receptor Status after Primary Systemic Therapy Is Associated with Poor Clinical Outcome in Patients with Breast Cancer. Cancer Research and Treatment : Official Journal of Korean Cancer Association, 50(4), 1418-1432.

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Biomarker Profiling in Breast Cancer

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For each case, basic biomarkers were reviewed on immunohistochemistry (IHC) slides. All IHC staining procedures were performed using BenchMark XT autostainer (Ventana Medical Systems, Tucson, USA) with an UltraView detection kit (Ventana Medical Systems). The following antibodies were used: ER (1:100; clone SP1; Labvision, Fremont, USA), PR (1:70; PgR 636; Dako, Carpinteria, USA), HER2 (ready to use; clone 4B5; Ventana Medical Systems), p53 (1:600; D07; Dako), and Ki-67 (1:250; MIB-1; Dako). ER and PR were considered positive if there were at least 1% positive tumor nuclei. For ER and PR, expression level was scored in 10% increments, and their Allred scores were calculated [13 (link)]. HER2 expression was scored according to 2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines [14 (link)]. For Ki-67 proliferation index, positive staining in 20% or more tumor cells was regarded as a high index. As biomarker status can change after neoadjuvant chemotherapy (especially PR and Ki-67 proliferation index) [15 (link)16 (link)], we used the biomarker data from the pre-treatment needle biopsy specimen in patients who received neoadjuvant chemotherapy.

Woo J.W., Chung Y.R., Ahn S., Kang E., Kim E.K., Kim S.H., Kim J.H., Kim I.A, & Park S.Y. (2019). Changes in Biomarker Status in Metastatic Breast Cancer and Their Prognostic Value. Journal of Breast Cancer, 22(3), 439-452.

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TNBC Immunohistochemistry and TILs Assessment

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Immunohistochemical analysis on BC specimens were performed at our Pathology Department and tumors were defined as TNBC if ER (Ventana, Clone SP1, pre-diluted) and PgR (clone 30-9; Ventana Medical Systems Inc, pre-diluted) IHC staining was ≤ 1%, together with 0/1 + score HER2 on IHC (clone 4B5; Ventana Medical Systems Inc, pre-diluted) and/or non-amplified on fluorescent in situ hybridization (FISH) for 2 + score HER2 cases. For each study patient, formalin-fixed paraffin-embedded (FFPE) tumor samples were retrieved from Institutional Pathology archives. Stromal TILs were assessed according to consensus guidelines^{7 (link),30 (link)} by one investigator (BF) who was blinded to clinical data. AR expression was measured by IHC, using an anti-androgen receptor (Clone SP107; Ventana Medical Systems Inc, pre-diluted) and the percentage of AR-positive nuclei was quantified. A cutoff of 10% or greater was used to define an AR-positive tumor^{31 (link)}. The IHC threshold (= 0 + or < 10% or ≥ 1 + and ≥ 10%) was used for AR.

Losurdo A., De Sanctis R., Fernandes B., Torrisi R., Masci G., Agostinetto E., Gatzemeier W., Errico V., Testori A., Tinterri C., Roncalli M, & Santoro A. (2020). Insights for the application of TILs and AR in the treatment of TNBC in routine clinical practice. Scientific Reports, 10, 20100.

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Breast Cancer Biopsy and Biomarker Analysis

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Tumor core biopsies were performed before NAC therapy commenced. ER, PgR, Ki-67 and HER2 were assessed by IHC. IHC staining for ER (Confirm anti-ER, rabbit monoclonal primary antibody; clone SP1, Ventana Medical Systems), PR (Confirm anti-PR, rabbit monoclonal primary antibody; clone 1E2; Ventana Medical Systems), ki-67 (mouse monoclonal primary antibody; clone MIB1; Origene), and HER2 (anti-HER2/neu, rabbit monoclonal primary antibody; clone 4B5; Ventana) was performed.
The percentage and intensity of nuclear staining with ER and PR were estimated, and nuclear staining of ≥ 1% of the invasive tumor nuclei was interpreted as positive, in accordance with 2010 ASCO/CAP guidelines. HER2 positivity refers to cases with an IHC score of 3 + or fluorescence in situ hybridization (FISH) amplification (by 2013 ASCO/CAP criteria) for IHC scores of 2+. Patients were separated into three groups for the purposes of our analysis according to ER expression level: ER-negative, ER low positive (ER staining 1-10%), and ER > 10% positive (ER staining > 10%). The ER expression status on the surgical specimen was recorded. The assessments were performed locally by two experienced pathologists.

Chen H.L., Huang F.B., Chen Q, & Deng Y.C. (2023). Impact of estrogen receptor expression level on response to neoadjuvant chemotherapy and prognosis in HER2-negative breast cancers. BMC Cancer, 23, 841.

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Multiplex IHC for Breast Cancer Biomarkers

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IHC staining for ER (mouse monoclonal, clone SP1, prediluted; Ventana Medical Systems), PR (mouse monoclonal, clone 1E2, prediluted; Ventana Medical Systems), HER2 (Her2/neu, rabbit monoclonal, clone 4B5, prediluted; Ventana Medical Systems), and Ki67 (rabbit monoclonal, clone SP6, prediluted; Ventana Medical Systems) was performed on 4 mm-thick tissue sections using a Ventana automated immune-stainer (Ventana Benchmark Ultra, Ventana Medical Systems). Pretreatment was performed with the onboard antigen retrieval method at high pH (8.4) using a cell conditioning 1 antigen retrieval buffer system at 95 °C (Ventana Medical Systems). The epitope retrieval time for antibodies was 32 minutes. The duration of incubation for primary antibodies was 24 minutes for ER, 8 minutes for PR and Her2/neu, and 10 minutes for Ki67. An indirect biotin-free system for the detection of various primary antibodies (Ultraview Universal Diaminobenzidine kit) was used for detection. The slides were then counterstained with hematoxylin (Harris).

Ping W., Xin R., Li Z., Yupeng C., Fangling S., Caihong R., Shun H, & Sheng Z. (2023). Effect of Surface Decalcification With Hydrochloric Acid on the Determination of Estrogen Receptor, Progesterone Receptor, Ki67, and Human Epidermal Growth Factor Receptor 2 Expressions in Invasive Breast Carcinoma Based on Immunohistochemistry and Fluorescence In Situ Hybridization. Applied Immunohistochemistry & Molecular Morphology, 31(4), 232-238.

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HER2 Immunohistochemistry Scoring Protocol

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For the study period, all IHC HER2 analysis tests were performed on 4-µm-thick sections of formalin-fixed, paraffin-embedded tissue using Ventana automation, the Ultra View detection kit, and a rabbit monoclonal antibody to HER2 (clone 4B5, prediluted, Ventana Medical Systems, Tucson, AZ). The intensity and the proportion of tumor cells stained were assessed on cytoplasmic membrane staining. All HER2 IHC results were interpreted and reviewed by a rotating group of six staff pathologists, all with specific experience in breast pathology.
Invasive breast cancers with 3+ uniform intense membrane staining in >10% of tumor cells were interpreted as HER2 IHC positive. An equivocal result was rendered when more than 10% of tumor cells showed weak-tomoderate cytoplasmic membrane staining. The results that did not fulfill the above criteria were considered negative.

Zare S., Rong J., Daehne S., Roma A., Hasteh F., Dell'Aquila M, & Fadare O. (2019). Implementation of the 2018 American Society of Clinical Oncology/College of American Pathologists Guidelines on HER2/neu Assessment by FISH in breast cancers: predicted impact in a single institutional cohort. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 32(11).

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Immunohistochemical Staining Protocol

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IHC was performed with a Ventana XT automated staining instrument. Antibodies recognizing the following targets were used: MutL homolog 1 (MLH1, ready to use, clone M1, Roche, Basel, Schweiz), MutS protein homolog 2 (MSH2, ready to use, clone G219-1129, Roche), MutS homolog 6 (MSH6, 1:100, clone 44, Cell Marque, Rocklin, CA), postmeiotic segregation increased 2 (PMS2, 1:40, clone MRQ28, Cell Marque), HER2 (ready to use, clone 4B5, Roche), EGFR (1:100, EP38Y, Abcam, Cambridge, UK), c-MET (ready to use, clone SP44, Roche), PTEN (1:100, clone 138G6, Cell signaling, Danvers, MA), and p53 (1:300, DO7, Novocastra, Newcastle, UK). Sections were deparaffinized using EZ Prep solution (Ventana Corporation, Tucson, AZ). CC1 standard (pH 8.4 buffer containing Tris/borate/EDTA) was used for antigen retrieval and blocked with inhibitor D (3% H₂O₂) for 4 min at 37°C. Slides were incubated with primary antibody for 40 min at 37°C, followed by a universal secondary antibody for 20 min at 37°C. Slides were incubated in streptavidin-horseradish peroxidase (SA-HRP) D for 16 min at 37°C, after which the substrate, 3,3′-diaminobenzidine tetrahydrochloride (DAB) H₂O₂, was added for 8 min, followed by hematoxylin and bluing reagent counterstaining at 37°C.

Kim H.S., Shin S.J., Beom S.H., Jung M., Choi Y.Y., Son T., Kim H.I., Cheong J.H., Hyung W.J., Noh S.H., Chung H., Park J.C., Shin S.K., Lee S.K., Lee Y.C., Koom W.S., Lim J.S., Chung H.C., Rha S.Y, & Kim H. (2016). Comprehensive expression profiles of gastric cancer molecular subtypes by immunohistochemistry: implications for individualized therapy. Oncotarget, 7(28), 44608-44620.

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