For intracellular cytokine staining (ICS), cells stimulated with indicated stimuli in the presence of GolgiStop (BD Biosciences) were stained for cell-surface markers, fixed and permeabilized using Cytofix/Cytoperm buffer (BD Biosciences) and then stained with fluorochrome-conjugated Abs to IFN-γ (XMG1.2; eBioscience; 1:300) and TNF-α (MP6-XT22; BD Biosciences; 1:300) using Perm/Wash buffer (BD Biosciences). The same ICS protocol was used for analysing Bcl-2 and CD107a expression with fluorochrome-conjugated Abs to Bcl-2 (BCL/10C4; Biolegend; 1:200) and CD107a (1D4B; eBioscience; 1:300). Intracellular staining for T-bet and Eomes expression was performed with Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer's instructions using fluorochrome-conjugated Abs to T-bet (4B10; 1:200) and Eomes (Dan11mag; all from eBioscience; 1:200). For intracellular staining for p-ERK, B6 SP cells treated with indicated stimuli were fixed with 2% paraformaldehyde at RT for 15 min, followed by permeabilization with ice-cold 90% methanol for 20 min on ice. After a washing step, cells were blocked with PBS containing 2% FBS and incubated with fluorochrome-conjugated Ab to p-ERK (Thr202/Tyr204; D13.14.4E; Cell Signaling Technology; 1:100), followed by repeated washes and continued incubation for 15 min on ice with fluorochrome-conjugated Abs to cell-surface markers.
Thr202 tyr204 d13.14.4e
Manufactured by Cell Signaling Technology
Sourced in United States
Thr202/Tyr204; D13.14.4E is a lab equipment product offered by Cell Signaling Technology. It is designed to detect phosphorylation of threonine 202 and tyrosine 204 residues in the human ERK1/2 protein.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 10c4, by BioLegend (1 mentions)
Dan11mag, by Thermo Fisher Scientific (1 mentions)
Mp6 xt22, by BD (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Golgistop, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Cd45 lca, by Agilent Technologies (1 mentions)
S 100 protein, by BioGenex (1 mentions)
Cd163, by Agilent Technologies (1 mentions)
Irf4 mum1, by Agilent Technologies (1 mentions)
2 protocols using thr202 tyr204 d13.14.4e
1
Intracellular Cytokine and Protein Staining
2
Immunohistochemical Analysis of Rosai-Dorfman Disease
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Immunohistochemical studies were performed using formalin-fixed, paraffin-embedded tissue sections as described previously [24] . The antibody panels for each case were variable according to lymphoma type and differences over time. The antibodies used included reagents specific for: CD1a and CD21 (Leica Biosystem, Newcastle, UK); CD2, CD3, CD4, CD7, CD8, CD20, CD30, CD45/LCA, CD68, CD163, Bcl-6, and MUM1/IRF4 (DAKO, Carpinteria, CA); CD5 (Labvision/Neomarkers, Fremont, CA); CD10, CD23, and Bcl-2 (Novocastra/Vision Biosystem, Benton Lane, Newcastle-upon-Tyne, UK); CD15 (Becton-Dickinson Biosciences, San Jose, CA); PAX-5 (Transduction Labs, San Diego, CA); Ki-67 (Ventana, Tucson, AZ); and S100 protein (BioGenex, Fremont, CA, USA).
Specifically for this study, in cases with available paraffin-embedded tissue sections we assessed for evidence of MAPK/ERK pathway activation in the foci of Rosai-Dorfman disease using antibodies for cyclin D1 (dilution 1:350; Labvision/Neomarkers) and phospho-p44/ 42 MAPK (Thr202/Tyr204) (D13.14.4E) and p-ERK (dilution 1:300, Cell Signaling, Danvers, MA). The latter antibody allows for assessment of nuclear and cytoplasmic phosphorylated p44 and p42 MAPK (Erk1 and Erk2). Results for cyclin D1 and p-ERK were considered positive if staining was present in >5% of Rosai-Dorfman disease histiocytes with moderate or strong intensity.
Specifically for this study, in cases with available paraffin-embedded tissue sections we assessed for evidence of MAPK/ERK pathway activation in the foci of Rosai-Dorfman disease using antibodies for cyclin D1 (dilution 1:350; Labvision/Neomarkers) and phospho-p44/ 42 MAPK (Thr202/Tyr204) (D13.14.4E) and p-ERK (dilution 1:300, Cell Signaling, Danvers, MA). The latter antibody allows for assessment of nuclear and cytoplasmic phosphorylated p44 and p42 MAPK (Erk1 and Erk2). Results for cyclin D1 and p-ERK were considered positive if staining was present in >5% of Rosai-Dorfman disease histiocytes with moderate or strong intensity.
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