Immunohistochemical staining of LMP1 (1:50, clone CS.1–4; Dako, Carpinteria, CA, USA), EBNA2 (1:50, clone PE2; Dako), BCL6 (1:10, clone PG-B6p; Dako), PRDM1 (1:50, clone 3H2E8; Santa Cruz Biotechnology, Dallas, TX, USA) and MUM1/IRF4 (1:100, clone MUM1p; Dako) were accomplished using the Bond III Autostainer (Leica Microsystems, Buffalo Grove, IL, USA). Formalin-fixed, paraffin-embedded tissues or cell block sections were first baked and deparaffinized. Antigens were then retrieved by heating the slides at 37 °C in Bond Enzyme solution (Leica Microsystems) for 10 min (for LMP1), and at 99–100 °C in Bond Epitope Retrieval Solution 2 for 20 (for EBNA2, PRDM1 and MUM1) or 30 min (for BCL6). Sections were then incubated sequentially with the endogenous peroxidase block, primary antibody, postprimary (equivalent to secondary antibody), polymer (equivalent to tertiary antibody), diaminobenzidine and hematoxylin for 5, 25, 15, 25, 10 and 5 min, respectively. Bond Polymer Define Detection (Leica Microsystems) was used for EBNA2 and MUM1, and Bond Polymer Refine Detection (Leica Microsystems) was used for LMP1, BCL6 and PRDM1. Finally, the stained slides were dehydrated and mounted in Cytoseal XYL (Richard-Allan Scientific, Kalamazoo, MI, USA).
Clone cs 1 4
Manufactured by Agilent Technologies
Sourced in United States
The Clone CS.1-4 is a laboratory equipment product designed for general laboratory use. It serves as a tool for performing various experimental procedures. The core function of the Clone CS.1-4 is to provide a versatile and reliable platform for researchers and scientists to conduct their work. No further details or interpretation of the intended use are provided.
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Lab products found in correlation
Bond polymer refine detection, by Leica (1 mentions)
Bond 3 autostainer, by Leica (1 mentions)
Bond epitope retrieval solution 2, by Leica (1 mentions)
Cytoseal xyl, by Thermo Fisher Scientific (1 mentions)
Clone mum1p, by Agilent Technologies (1 mentions)
Alexa fluor 488 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Antifade mounting medium containing 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Benchmark xt, by Roche (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Bond polymer refine detection kit, by Leica (1 mentions)
Bond max, by Leica (1 mentions)
5 protocols using clone cs 1 4
1
Immunohistochemical Characterization of B-cell Markers
2
Immunohistochemistry for EBV Proteins in Multiple Sclerosis
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EBER in situ hybridization was performed using the PNA probe, which hybridizes with both EBER1 and EBER2, and the detection kit from DakoCytomation (Glostrup, Denmark), as described previously (35 (link), 37 (link)).
Immunohistochemical stainings for EBV latent (EBNA2) and lytic (BZLF1) proteins and double immunofluorescence stainings for LMP1 and the B-cell marker CD79a and for BZLF1 and the plasma cell marker IgA, IgG, and IgM were performed in sections from paraformaldehyde (PFA)-fixed frozen brain tissue blocks of three MS donors analyzed in this study (MS79, MS92, and MS121), as previously described (19 (link), 35 (link)– (link)38 (link)). For double immunofluorescence staining for LMP1 and LMP2A, brain sections were incubated with anti-LMP2A rat MAb (1:50; clone TP4E11; Ascenion, Munich, Germany) and anti-LMP1 mouse MAb (1:100; clone CS.1-4; DakoCytomation) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin overnight at 4°C, and, after washing with a mixture of Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen, Eugene, OR) and tetramethylrhodamine (TRITC)-conjugated donkey anti-rat Ig (Jackson ImmunoResearch Laboratories, Cambridgeshire, UK), both diluted 1:300 in PBS containing 3% normal donkey serum. After further washings, sections were mounted with antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).
Immunohistochemical stainings for EBV latent (EBNA2) and lytic (BZLF1) proteins and double immunofluorescence stainings for LMP1 and the B-cell marker CD79a and for BZLF1 and the plasma cell marker IgA, IgG, and IgM were performed in sections from paraformaldehyde (PFA)-fixed frozen brain tissue blocks of three MS donors analyzed in this study (MS79, MS92, and MS121), as previously described (19 (link), 35 (link)– (link)38 (link)). For double immunofluorescence staining for LMP1 and LMP2A, brain sections were incubated with anti-LMP2A rat MAb (1:50; clone TP4E11; Ascenion, Munich, Germany) and anti-LMP1 mouse MAb (1:100; clone CS.1-4; DakoCytomation) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin overnight at 4°C, and, after washing with a mixture of Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen, Eugene, OR) and tetramethylrhodamine (TRITC)-conjugated donkey anti-rat Ig (Jackson ImmunoResearch Laboratories, Cambridgeshire, UK), both diluted 1:300 in PBS containing 3% normal donkey serum. After further washings, sections were mounted with antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).
3
Immunohistochemical Profiling of Oncoproteins
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MHC-I, MHC-II, PDL1, LMP1, MTAP protein expressions were determined on formalin-fixed, paraffin-embedded sections by immunohistochemical staining. After de-waxing, the sections were subjected to antigen retrieval and staining by the automated slide processing system BenchMark XT (Ventana Medical System Inc., Tucson, AZ, USA) as previously published8 (link),35 (link). The primary antibody used in this study was anti-LMP1 mouse monoclonal antibody (diluted 1:1000, clone CS.1-4, Dako, Agilent Technologies, USA), anti-PDL1 (22C3, Dako, Agilent Technologies, USA), anti-HLA Class I A/B/C (diluted 1:1000, clone ECR8-5, ab7038, Abcam, USA), anti-MHC Class II (diluted 1:2000, clone 6C6, ab55152, Abcam, USA), anti-MTAP (diluted 1:400; 4158, Cell Signaling, USA), anti-Involucrin (diluted 1:100, clone SY5, MA5-11803, Invitrogen, USA) and p53 (diluted 1:100, clone DO-1, SC-126, Santa Cruz, USA).
4
Western Blotting Antibody Detection
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Western blotting was performed as described previously10 (link) with the following antibodies: anti-LMP1 (1:250; Dako, clone CS1-4, Glostrup, Denmark), anti-α-syn (EP1646Y; 1:10,000; Abcam ab51252, Cambridge, UK), and anti-β-actin (1:5,000; Sigma A2228, Munich, Germany). Between antibody exposures, the membrane was stripped to remove previous immunoreactivity (Thermo Fisher Scientific 46430, Waltham, MA). Blots shown are representative of at least 3 separate experiments.
5
Immunohistochemical Assessment of HPV and EBV
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The p16 INK4a expression was assessed using an autostaining system (Leica Bond-Max), the BOND Polymer Refine Detection Kit (both from Leica Microsystems GmbH), and a monoclonal mouse antibody (Roche Diagnostics GmbH, Mannheim, Germany). The stained tumor areas were dichotomized as follows: adopted from Schauer et al., we used an immunostaining score comprised of intensity and a stained tumor area that had values between 0 and 7 [23] . To perform the statistical analysis, we set a cut off at 4 and divided the samples into positive and negative.
The HPV activity in a cell could be shown by determining the level of the p16 expression [24] . Because p16 overexpression could occur independently from HPV infection, HPV patients with a positive HPV status and a high level of p16 expression were considered genuinely HPV positive. In this study, the cells that were both p16 INK4a positive and HPV positive were considered genuinely HPV positive.
To measure EBV activity we stained the SNSCC sections with a monoclonal mouse antibody against LMP1 (Clone CS 1-4, DAKO Ò ). Staining was performed on a manual base. Evaluation of stained areas was done accordingly to p16 INK4a . The cut off was set at 2. Samples with both, EBV ISH positivity and LMP1 expression were considered genuinely EBV positive.
The HPV activity in a cell could be shown by determining the level of the p16 expression [24] . Because p16 overexpression could occur independently from HPV infection, HPV patients with a positive HPV status and a high level of p16 expression were considered genuinely HPV positive. In this study, the cells that were both p16 INK4a positive and HPV positive were considered genuinely HPV positive.
To measure EBV activity we stained the SNSCC sections with a monoclonal mouse antibody against LMP1 (Clone CS 1-4, DAKO Ò ). Staining was performed on a manual base. Evaluation of stained areas was done accordingly to p16 INK4a . The cut off was set at 2. Samples with both, EBV ISH positivity and LMP1 expression were considered genuinely EBV positive.
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