Formaldehyde-fixed, paraffin-embedded tissue sections of 3µm thickness from tumor resection or biopsy were used for immunohistochemistry. Monoclonal mouse anti-human primary antibody for p16INK4A (1:50; clone: G175-405; BD Biosciences; Cat. No. 550834), HLA-ABC (1:100; clone: EMR8-5; Abcam; Cat. No. AB70328), CD20 (1:250; clone L26; Dako; Cat. No. M0755) and MUM1 (1:1000; clone: MUM1p; Dako; Cat. No. M725929-2) were applied. Detection of primary antibodies was achieved by secondary goat anti-mouse antibody conjugated with a HRP-labelled polymer (Dako EnVision+) and peroxidase activity was visualized by diaminobenzidine tetrahydrochloride. Hematoxylin was used as nuclear counterstaining. Detection of HLA-ABC expression in ≥ 70% of tumor cells counted as high MHC-I expression, strong cytoplasmic and nuclear staining was regarded positive for p16INK4A expression.
G175 405
Manufactured by BD
Sourced in United States
The G175-405 is a laboratory instrument designed for general purpose use. It features a compact and durable construction, making it suitable for a variety of laboratory settings. The core function of this equipment is to perform basic laboratory tasks, though the specific intended use is not provided.
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Lab products found in correlation
Clone l26, by Agilent Technologies (1 mentions)
Mum1p, by Agilent Technologies (1 mentions)
Emr8 5, by Abcam (1 mentions)
Actin 1 19, by Santa Cruz Biotechnology (1 mentions)
H 435, by Santa Cruz Biotechnology (1 mentions)
Envision system hrp dab kit, by Agilent Technologies (1 mentions)
Y1404, by Agilent Technologies (1 mentions)
Gradient gel, by Bio-Rad (1 mentions)
Ecl prime reagent, by Cytiva (1 mentions)
Pt link module, by Agilent Technologies (1 mentions)
Autostainer, by Agilent Technologies (1 mentions)
9 protocols using g175 405
1
Immunohistochemical Analysis of Tumor Markers
2
Antibody Panel for Cell Cycle Regulation
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Antibodies. The following antibodies were used in this study. Skp2 (Rabbit polyclonal H-435, Santa Cruz # sc-7164, 1:500), Skp2 (Mouse monoclonal A2, Santa Cruz #sc-74477 1:250), p27 (Mouse monoclonal, BD Transduction laboratoriesn#610242,1 in 250), p27 (rabbit polyclonal, C19 # sc-528, SantaCruz), p21 Mouse (Mouse monoclonal anti-human Cip1, BD Transduction laboratories, #610234), 1 in 250) p57 (C-20, Santa Cruz,sc-1040) p16 INK4a (Mouse monoclonal, G175-405, Pharmingen # 13251A, 1in 100), p15 Ink4b (Rabbit polyclonal, K-18, Santa Cruz, # sc-613) Cyclin E (mouse monoclonal HE12, Santa Cruz), Cyclin A (Polyclonal rabbit serum, produced in Hengst laboratory), PSTAIRE (Murine cell supernatant, Cdk4 (Rabbit Polyclonal, C-22, Santa Cruz, sc-260), Tubulin (mouse monoclonal, b-tubulin AA2 Sigma-Aldrich T8328), Cdh1/Fizzy related (homemade, rabbit 1 in 100), GAPDH mouse monoclonal (6C5, Calbiochem 1in 1000), Actin (I-19, Santa Cruz), GFP (Mouse Mixture of two monoclonal antibodies (7.1 and 13.1) Roche, # 11814460001, 1in 1000) Flag (mouse monoclonal, M2 Sigma Aldrich #F3165).
3
Immunohistochemical detection of p16INK4a and pan-CK
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FFPE tissue sections were deparaffinized, rehydrated, antigen retrieved using Proteinase K, and blocked in sheep serum and BSA solution as described above in ISH section. p16INK4a antigen was retrieved by boiling tissue sections in Tris-EDTA (pH 9) in a microwave oven for 15 minutes. Thereafter, tissue sections were incubated with monoclonal mouse anti-human primary antibody (pan-CK 1:800, Clone MNF116, Agilent Dako; p16INK4a 1:1000, G175-405, BD Pharmingen, NewYork, NJ, USA) at RT for 1 h. A Dako Envision+ System-HRP (DAB) kit (K4007; Agilent Dako) was used for the subsequent steps. Tissue endogenous peroxidase activity was blocked with peroxidase block for 5 min. Thereafter, sections were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min and visualized with diaminobenzidine (DAB) substrate at RT. Tissues were counterstained with fast red and mounted with Pertex.
4
HPV Testing in Tumor Samples
5
Immunohistochemical Detection of p16 and HPV DNA
6
Immunohistochemical Analysis of p16INK4a
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IHC for p16INK4a (G175-405, BD Pharmingen) expression was performed as previously described [16 , 22 (link)]. Sections of p16INK4a cervical carcinoma were used as positive controls.
P16INK4a expression was scored according to staining intensity and percentage positive tumor cells. Tumors were classified as nuclear p16INK4a expressing, cytoplasmic p16INK4a expressing, and p16INK4a negative groups. The nuclear p16INK4a expressing group was defined as nuclear p16INK4a expression in >10% of the carcinoma cells. The cytoplasmic p16INK4a expressing group was defined as only cytoplasmic p16INK4a expression in >10% of carcinoma cells. The p16INK4a negative group was defined as <10% p16INK4a nuclear and/or cytoplasmic staining of carcinoma cells [23 (link), 24 (link)]. HPV status was determined by HPV based GP5+/6+ PCR as previously described [22 (link)].
P16INK4a expression was scored according to staining intensity and percentage positive tumor cells. Tumors were classified as nuclear p16INK4a expressing, cytoplasmic p16INK4a expressing, and p16INK4a negative groups. The nuclear p16INK4a expressing group was defined as nuclear p16INK4a expression in >10% of the carcinoma cells. The cytoplasmic p16INK4a expressing group was defined as only cytoplasmic p16INK4a expression in >10% of carcinoma cells. The p16INK4a negative group was defined as <10% p16INK4a nuclear and/or cytoplasmic staining of carcinoma cells [23 (link), 24 (link)]. HPV status was determined by HPV based GP5+/6+ PCR as previously described [22 (link)].
7
Western Blot Analysis of Cell Cycle Proteins
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Details about WB analysis conditions are reported in Supplementary Table 1 . In brief, after cell lysis, equal amount of proteins was loaded on a 4–20% gradient gel (BioRad) and separated by SDS-PAGE. The membrane was further probed with antibodies against CDK4 (EPR4513-32-7, abcam, dilution 1:1000) and CDKN2A (G175-405, BD Pharmingen, San Jose, CA, USA, dilution 1:500).
It is also known that CDK4, along with CDK6, activated by binding to D-type cyclins, is responsible for the G1-S phase transition in the cell cycle by phosphorylating and inhibiting the retinoblastoma (RB) protein and the related proteins p107/RBL1 and p130/RBL2 (36 (link)). Moreover, CDK4/6 inhibitors have been reported to reduce cell viability in ACC cell lines despite lack of pRb expression (28 (link), 31 (link)) p130/RBL2 being already reported to be expressed in the NCI-H295R cell line (28 (link)). Thus, we investigated RB and p130/RBL2 protein expression in both cell lines. Signal detection was achieved by incubation with appropriate HRP-labeled secondary antibodies and Amersham ECL Prime reagent visualizing the protein-antibody complex by enhanced chemiluminescence.
It is also known that CDK4, along with CDK6, activated by binding to D-type cyclins, is responsible for the G1-S phase transition in the cell cycle by phosphorylating and inhibiting the retinoblastoma (RB) protein and the related proteins p107/RBL1 and p130/RBL2 (36 (link)). Moreover, CDK4/6 inhibitors have been reported to reduce cell viability in ACC cell lines despite lack of pRb expression (28 (link), 31 (link)) p130/RBL2 being already reported to be expressed in the NCI-H295R cell line (28 (link)). Thus, we investigated RB and p130/RBL2 protein expression in both cell lines. Signal detection was achieved by incubation with appropriate HRP-labeled secondary antibodies and Amersham ECL Prime reagent visualizing the protein-antibody complex by enhanced chemiluminescence.
8
HPV Detection in Oropharyngeal Tumors
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Since HPV status was not considered in the first paper, this was currently assessed [12] . For all patients with oropharyngeal tumors, formalin-fixed, paraffin-embedded (FFPE) tissue was centralized (University Hospital of Leuven) for HPV-status determination. HPV testing was performed using a previously validated algorithm using p16 immunohistochemistry (IHC) followed by HPV-polymerase chain reaction (PCR) [13] [14] [15] . A tumor was regarded as HPV related when both p16 IHC as well as HPV-PCR were positive.
For p16 IHC a purified mouse anti-human p16 antibody (G175-405, BD Pharmigen) was used. Sections were scored as p16 positive when clear p16 immunoreactivity was seen in at least 50% of cells [14] . DNA was extracted from PPFE sections using the QIAamp DNA FFPE Tissue kit. Concentration and purity where then defined with spectrophotometry. HPV status was determined with a PCR reaction using the GP5+/6+ primer set.
For p16 IHC a purified mouse anti-human p16 antibody (G175-405, BD Pharmigen) was used. Sections were scored as p16 positive when clear p16 immunoreactivity was seen in at least 50% of cells [14] . DNA was extracted from PPFE sections using the QIAamp DNA FFPE Tissue kit. Concentration and purity where then defined with spectrophotometry. HPV status was determined with a PCR reaction using the GP5+/6+ primer set.
9
Oropharyngeal Cancer HPV Screening
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All patients diagnosed with OPSCC between 2004 and 2012 were included in the study. The primary inclusion criterion was availability of formalin-fixed, paraffinembedded tumor tissue. For each patient, we collected the following data: identification number, date of birth, gender, tobacco and alcohol use (as noted in the medical records), oropharyngeal subsite, tumor-nodemetastasis (TNM) class, and tumor stage (based on American Joint Commission on Cancer [AJCC] Cancer Staging Manual, 7th edition), type of therapy, start and end dates of therapy, dates of local or regional relapses, date of distant recurrence, date of death, and date of last follow-up.
Tumor samples and laboratory studies For all patients, formalin-fixed, paraffin-embedded tissue was used to determine HPV status. HPV testing was performed with p16 IHC on 5-mm tissue sections. A DAKO PT link module and a DAKO autostainer were used with an automated protocol for deparaffinization, antigen retrieval, and IHC. A purified mouse antihuman p16 antibody (G175-405, BD Pharmigen) was used for p16 IHC. Sections were scored as HPV-positive when a clear, p16 immunoreactive signal was detected in at least 50% of cells. 8
Tumor samples and laboratory studies For all patients, formalin-fixed, paraffin-embedded tissue was used to determine HPV status. HPV testing was performed with p16 IHC on 5-mm tissue sections. A DAKO PT link module and a DAKO autostainer were used with an automated protocol for deparaffinization, antigen retrieval, and IHC. A purified mouse antihuman p16 antibody (G175-405, BD Pharmigen) was used for p16 IHC. Sections were scored as HPV-positive when a clear, p16 immunoreactive signal was detected in at least 50% of cells. 8
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