The following primary antibodies were used in this study: mouse anti-EGFR (1:10, Novacastra, UK), mouse anti-HER-2 (1:150, Insight Biotechnology, UK), mouse anti-HER-3 RTJ.2 (1:50, Insight Biotechnology, UK), rabbit anti-HER-4 (1:20, Fisher Scientific, UK), rabbit anti-EGFRvIII (1:150, Bioss Antibodies, UK), mouse anti-IGF-1R (4 ug/mL, Merck Millipore, UK), mouse anti-c-MET (1:500, Novacastra, UK) and mouse anti-CD44 (1:40, DAKO, UK). Formalin-fixed paraffin-embedded (FFPE) sections of tumour specimens (3 μM) were cut in serial sections, and then subjected to antigen retrieval and incubation with primary antibodies as described previously by Khelwatty and colleagues [22 (link)]. The optimisation for HER-3 and IGF-1R staining was conducted using the MCF-7 (HER-3+) breast cancer cell line and tumour section of the placenta respectively. Staining of the slides was carried out on a Ventana Benchmark Ultra autostainer with the Ultra View DAB kit (Roche, UK). Following this, all slides were rehydrated and counterstained with haematoxylin, mounted and cover slipped.
Ultraview dab kit
Manufactured by Roche
Sourced in United States, United Kingdom
The UltraView DAB kit is a labeling kit used in immunohistochemistry. It provides a sensitive detection system for visualizing target antigens in tissue samples. The kit utilizes a diaminobenzidine (DAB) chromogen to generate a brown staining reaction, allowing for the identification and localization of specific proteins or cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Benchmark ultra, by Roche (8 mentions)
Superfrost slide, by Thermo Fisher Scientific (4 mentions)
Rabbit anti her 4, by Thermo Fisher Scientific (2 mentions)
Cc1 mild protocol, by Roche (2 mentions)
Monoclonal anti p cadherin antibody clone 56, by BD (2 mentions)
Cd31 jc70 clone, by Cell Marque (2 mentions)
Benchmark ultra system, by Roche (2 mentions)
Ventana benchmark ultra, by Roche (1 mentions)
Benchmark xt system, by Roche (1 mentions)
Ventana benchmark immunostainer, by Roche (1 mentions)
Ez prep, by Roche (1 mentions)
Flex ihc microscope slides, by Agilent Technologies (1 mentions)
Ck7 clone ov tl 12 30, by Agilent Technologies (1 mentions)
Erbb2 clone 4b5, by Roche (1 mentions)
Er clone sp1, by Roche (1 mentions)
Ema clone e29, by Agilent Technologies (1 mentions)
Bcl2 clone 124, by Agilent Technologies (1 mentions)
Discovery xt, by Roche (1 mentions)
16 protocols using ultraview dab kit
1
Immunohistochemical Analysis of Receptor Proteins
2
Immunohistochemical Analysis of HER Family in FFPE Tumor Sections
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Formalin fixed paraffin-embedded (FFPE) sections of tumour specimens (3 µM) were cut in serial sections and were stained using the following primary antibodies mouse anti-EGFR (1∶10, Novacastra, UK), mouse anti-HER-2 (1∶150, Insight biotechnology, UK), mouse anti-HER-3 (1∶20, Novacastra, UK) and rabbit anti-HER-4 (1∶20, Fisher Scientific, UK). Following antigen retrieval, tumour sections were incubated with primary antibodies anti EGFR, HER-3 and HER-4 for 60 minutes and HER-2 for 32 minutes. Protocol optimisation was carried using established HER positive cancer cell line pellets, namely the EGFR overexpressing human colorectal cancer cell line DiFi, which was kindly provided by Dr Z Fan (MD-Anderson Cancer Centre, USA), the HER-2 overexpressing human breast carcinoma cell line SKBR3 (HER-2), and the HER-3 and HER-4 positive human breast carcinoma cell line MCF-7 as described previously [24] (link). Staining was carried out on a Venatana Benchmark XT autostainer with the ultraView DAB kit (Roche, UK). Finally, all slides were rehydrated and counterstained with haematoxylin, mounted and cover slipped.
3
Immunohistochemical Profiling of CNB Specimens
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For each CNB specimen of cases, IHC staining for CK5 and p63 was performed. IHC staining for CK5 was conducted using antibodies against CK5 (XM26, Leica Biosystems, Newcastle upon Tyne, UK) with 1:200 antibody dilution and a detection kit (Ivew DAB kit, Ventana, Tucson, AZ, USA). IHC staining for p63 was performed using antibodies against p63 (BC4A4, Biocare Medical, Pacheco, CA, USA) with 1:100 antibody dilution and a detection kit (Ultraview DAB kit, Ventana). According to the manufacturer’s protocol, all the procedures of IHC staining were processed by a Ventana BenchMark XT system (Ventana).
4
Immunohistochemical Analysis of FFPE Tissue
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Formalin‐fixed paraffin‐embedded (FFPE) tissue specimens were prepared and evaluated in the Institute of Pathology of the Hannover Medical School. Immunohistochemistry was performed on whole slide sections (1 µm) of FFPE tissue blocks on a Benchmark Ultra (Ventana, Tucson, USA) automated stainer. The CC1 mild protocol (Ventana) was used for antigen retrieval and the ultraView DAB kit (Ventana) for signal detection. Antibodies are detailed in supplementary material, Table S1. Written informed consent for case presentation was obtained by the corresponding patient. All analyses were conducted in accordance with the guidelines of the local ethics committee (MHH, Hannover, Germany).
5
Immunohistochemical Evaluation of Cadherins
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For immunohistochemistry, 1 µm thick sections of FFPE tissue blocks or TMAs were mounted on superfrost slides (Thermo Fisher Scientific, Rockford, USA). Next, slides were deparaffinized and rehydrated conventionally and were subjected to immunohistochemical staining using a Benchmark Ultra (Ventana, Tucson, USA) automated stainer. The CC1 mild program was used for antigen retrieval and the ultraView DAB kit (Ventana) for signal detection. Antibodies used for immunohistochemistry included the monoclonal anti-E-cadherin antibody ECH-6 (1:100, Zytomed, Berlin, Germany), the monoclonal anti-beta-catenin antibody clone 14 (1:75, BD Transduction Laboratories, Franklin Lakes, USA) the monoclonal anti-P-cadherin antibody clone 56 (1:100, BD Transduction Laboratories), the monoclonal anti-N-cadherin antibody 3B9 (1:100, Thermo Fisher Scientific, Rockford, USA) and the monoclonal anti-R-cadherin antibody D-9 (1:50, Santa Cruz, Santa Cruz, USA) Further antibodies are described in Supplementary data Table 1 (Supplementary data Table 1 ). Membranous P-cadherin immunoreactivity was scored using an immunoreactivity score (IRS) as described by Remmele and Stegner [22 (link), 23 (link)]. Immunohistochemistry scoring was discussed on a multi-headed microscope until consensus was achieved among ≥3 pathologists.
6
Immunohistochemistry for FFPE Tissue Sections
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For immunohistochemistry, 1 μm‐thick sections of FFPE tissue blocks were mounted on superfrost slides (Thermo Fisher Scientific, Rockford, IL, USA). Next, slides were deparaffinized and rehydrated conventionally and were subjected to immunohistochemical staining on a Benchmark Ultra (Ventana, Tucson, AZ, USA) automated stainer. The CC1 mild program was used for antigen retrieval, and the ultraView DAB kit (Ventana) was used for signal detection. Antibodies and scoring methods used for immunohistochemistry are summarized in supplementary material, Table S1 .
7
Automated IHC Staining of FFPE TMAs
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Batch-based immunohistochemical staining was performed on whole slide sections (1 μm) of FFPE TMAs on a Benchmark Ultra (Ventana) automated stainer. The CC1 mild protocol (Ventana) was used for antigen retrieval and the monoclonal anti-CD45 antibody (clone: 2B11+PD7/26, Dako, Glostrup, Denmark) for immunodetection. Development of the immune reaction was achieved with the ultraView DAB kit (Ventana). For counterstaining, sections were incubated in modified Gill’s hematoxylin (48%, Ventana, ready-to-use) and 0.1 M Li2CO3/0.5 M Na2CO3 (bluing reagent, Ventana, ready-to-use) for 8 min and 4 min, respectively.
8
MBOAT7 Immunostaining in Chronic Hepatitis C
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Immunostaining for MBOAT7 was performed on liver biopsies of CHC patients. Formalin-fixed, paraffin-embedded 4 μm sections were stained using a Ventana Benchmark Immunostainer (Ventana Medical Systems, Inc, Arizona, USA). Slides were incubated with a dilution of 1:10 of rabbit polyclonal antibody (Ab) specific for Human MBOAT7 (SIGMA ALDRICH). Negative controls where the primary antibody was excluded confirmed the specificity of immunostaining. Detection was performed using Ventana's Ultra View DAB kit (Roche/Ventana 05269806001). The pathologist (D.M.) evaluated the MBOAT7 immunostaining semi-quantitatively in a blinded fashion regarding any of the histological and clinical characteristics of the patients. The extent of staining was scored according to its amount and intensity using a 4-point scoring system as follows: 0=no staining; 1=positive staining in <33% of cells; 2=33–66% of positive cells; and 3=positive staining in more than 66% of cells.
9
Immunohistochemical Analysis of p16 Expression
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The TMAs were fabricated, including 5 mm oropharyngeal samples. The tissue microarrays were introduced into Benchmark Ultra equipment (Ventana, Medical systems Inc., Tucson, AZ, USA), and Tris-Borate-EDTA (TBE) buffer pH 8.0 was added for 92 min for antigen recovery. The microarrays were incubated at 37 °C in a humid chamber with the Cintec p16 antibody (Ventana, Medical systems Inc., Tucson, AZ, USA). The revealing of the antibody was carried out with the UltraView Dab Kit (Ventana, Medical systems Inc., Tucson, AZ, USA). The results for p16 IHC were interpreted by an experienced pathologist and were expressed as negative and positive according to the guidelines of the College of American Pathologists (positive with ≥70% nuclear and cytoplasmic staining) [24 (link)].
10
Immunohistochemical Evaluation of Neuroendocrine Tumor
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Formalin-fixed paraffin-embedded tissue samples were cut at 4 μm thick sections and mounted on coated slides (Dako Flex IHC microscope slides). To remove the paraffin EZ-prep from Ventana® was used. Afterwards slides were pretreated with Ventana CC1 (cell conditioning pH8.5) for 64 min. and incubated with antibody 32 min./36° diluted 1:100 in Dako antibodydiluent S2022 In Ventana Benchmark Ultra. The reaction was visualized by using Ventana Ultra View DAB-kit. Afterwards the sections were counterstained with Ventana Haematoxylin for 8 min.
Twenty hot spot areas (i.e. 20 areas within the tumor with a high count of immunoreactive tumor nuclei) were estimated and the mean percentage of Ki-67 cells calculated. An experienced neruroendocrine tumor pathologist with no knowledge of patient related prognostic information calculated the Ki-67 index.
To perform immunohistochemical idenfication of chromogranin A in tumor tissue we used Polyclonal Rabbit Anti-Human Chromogranin A (Dako Denmark A/S®, Glostrup, Denmark, code A0430). To identify positivity of synaptophysin and CD56, Monoclonal Mouse Anti Human Synaptophysin (Clone Snp 88, BioGenex Laboratories Inc. ®, Fremont, U.S.A., code MU363) and Monoclonal Mouse Anti-Human CD56 (Clone 1B6, NovoCastra®, Trichem, Skanderborg, Denmark, code NCL-CD56-1B6) were used, respectively.
Twenty hot spot areas (i.e. 20 areas within the tumor with a high count of immunoreactive tumor nuclei) were estimated and the mean percentage of Ki-67 cells calculated. An experienced neruroendocrine tumor pathologist with no knowledge of patient related prognostic information calculated the Ki-67 index.
To perform immunohistochemical idenfication of chromogranin A in tumor tissue we used Polyclonal Rabbit Anti-Human Chromogranin A (Dako Denmark A/S®, Glostrup, Denmark, code A0430). To identify positivity of synaptophysin and CD56, Monoclonal Mouse Anti Human Synaptophysin (Clone Snp 88, BioGenex Laboratories Inc. ®, Fremont, U.S.A., code MU363) and Monoclonal Mouse Anti-Human CD56 (Clone 1B6, NovoCastra®, Trichem, Skanderborg, Denmark, code NCL-CD56-1B6) were used, respectively.
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