Transverse kidney sections were dehydrated and paraffin-embedded, 4-μm sections were cut on a rotary microtome. Staining procedures were performed on the Ventana Benchmark Ultra automatic stainer. For antigen retrieval, deparaffinized sections were incubated with Cell Conditioning 1 medium (Ventana Medical Systems, Tucson, AZ, United States) at 100°C for 32 min. For immunolabeling, slides were incubated in the presence or absence of a polyclonal rabbit anti-CD3ε antibody (1:600, Novus Biologicals, Abingdon, United Kingdom) at 36°C for 32 min, followed by an 8-min incubation with a secondary antibody (Optiview HQ Universal Linker) at 36°C. Detection and visualization was done by subsequent 8-min incubations at 36°C with Optiview HRP Multimer and diaminobenzidine (all from Ventana Medical Systems), after which slides were counterstained with hematoxylin. A rat spleen sample was used as positive control. CD3+ cells were counted in 20 randomly selected 400 μm × 200 μm fields in cortex, outer and inner medulla by an investigator blinded to treatment allocation (D.S.). Results are expressed as positive cell counts per mm2.
Optiview hrp multimer
Manufactured by Roche
Optiview HRP Multimer is a laboratory equipment product by Roche. It is designed to enhance the detection of specific target molecules in various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Benchmark ultra, by Roche (1 mentions)
Benchmark xt, by Roche (1 mentions)
Opti view hq linker, by Roche (1 mentions)
Anti idh1 r132h antibody h09, by Dianova (1 mentions)
Ventana optiview amplification kit, by Roche (1 mentions)
Hpa001906, by Merck Group (1 mentions)
Cell conditioning 1 buffer, by Roche (1 mentions)
Optiview hq universal linker, by Roche (1 mentions)
3 protocols using optiview hrp multimer
1
Quantification of Renal CD3+ Cells
2
Immunohistochemical Analysis of NF1 Expression
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IHC for NF1 was performed using the IHC-plus™ polyclonal rabbit anti-human NF1 antibody (1:500; cat. no. LS-B14758; LifeSpan BioSciences, Inc.). All staining procedures were performed using a BenchMark XT automatic immunostaining device (Ventana Medical Systems) according to the manufacturer's protocol. Briefly, antigen retrieval was done by boiling sections using Cell Conditioning 1 buffer (cat. no. 950-124; Ventana Medical Systems, Inc.) for 32 min at 95°C. The sections were incubated with the anti-NF1 antibody for 16 min at 37°C in the automatic immunostainer. Signals were visualized using the Ventana OptiView DAB IHC Detection kit (cat. no. 06396500001; Ventana Medical Systems, Inc.): OptiView HQ Linker for 8 min at 37°C, Optiview HRP Multimer for 8 min at 37°C, OptiView H2O2/DAB for 8 min at 37°C and OptiView Copper for 4 min at 37°C. All specimens were reviewed and scored semi-quantitatively by a pathologist (JK) who gave a score ranging from 0 to 3. Since the NF1 staining was mostly diffuse, the four-tier scoring system was based on the average staining intensity (1, weak; 2, moderate; 3, strong). In addition, 0 was given only when NF1 expression was negligible in all tumor cells. NF1 was regarded to be lost if cytoplasmic staining was absent in tumor cells in the presence of intact expression in the internal non-neoplastic control cells.
3
Immunohistochemical Characterization of Gliomas
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Immunohistochemistry was conducted on 4 µm thick formalin-fixed, paraffin-embedded (FFPE) tissue sections mounted on StarFrost Advanced Adhesive slides (Engelbrecht, Kassel, Germany) followed by drying at 80°C for 15 min. Immunohistochemistry was performed on a BenchMark Ultra immunostainer (Ventana Medical Systems, Tucson, AZ, USA). Sections were stained with anti-IDH1-R132H antibody H09 (Dianova, Hamburg, Germany) as previously described [3] . ATRX immunohistochemistry was performed as previously described [19] . In brief, after deparaffinization, slides were pretreated at 95°C in Cell Conditioning 1 buffer (Ventana) for 90 minutes. The sections were incubated with primary antibody HPA001906 (Sigma-Aldrich, St. Louis, MO; USA) diluted 1:200 for 2 hours. Standard Ventana signal amplification was used. For BRAFV600E staining V600E-specific clone VE1 was used. After pretreatment with cell conditioner 1 (pH 8) for 64 min, sections were incubated with VE1 hybridoma supernatant (monoclonal, dilution 1:5) at 37°C for 32 min. Antibody incubation was followed by OptiView HQ Universal Linker for 12 min, incubation with OptiView HRP Multimer for 12 min, signal amplification including the Ventana OptiView Amplification Kit (Ventana, catalogue number 760-099).
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