Cryosections from colonic biopsies of IBD patients were used for immunohistochemistry. Tissue sections were fixed in 4% paraformaldehyde, followed by sequential incubation with avidin/biotin- (Vector Laboratories), and protein-blocking reagent (Roth) to suppress unspecific background staining. Sections were incubated with primary antibodies specific for human CD3 (rat anti-human CD3, Bio-Rad, Cat. MCA1477), human CD4 (rat anti-human CD4, Bio-Rad, Cat. MCA484G), and FITC-labeled vedolizumab (Entyvio®, Takeda). Furthermore, sections were incubated with an isotype matched control antibody as negative controls. Subsequently, samples were incubated with Alexa 647 conjugated secondary antibodies. Nuclei were counterstained with DAPI before final analysis by confocal microscopy (Leica SP8 Microscope). Positive cells in 6–10 high power fields (HPFs) were subsequently counted in all patients. In some images, an inset of a higher magnification was included to better identify the stained nuclei.
Entyvio
Manufactured by Takeda Pharmaceuticals
Entyvio is a laboratory equipment product manufactured by Takeda Pharmaceuticals. It is designed for use in research and clinical settings, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Sp8 microscope, by Leica camera (1 mentions)
Protein blocking reagent, by Carl Roth (1 mentions)
Avidin biotin, by Vector Laboratories (1 mentions)
Humira, by Abbvie (1 mentions)
Macsquant16, by Miltenyi Biotec (1 mentions)
Alexa fluor antibody labeling kit, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 506, by Thermo Fisher Scientific (1 mentions)
Vioblue, by Miltenyi Biotec (1 mentions)
Macsquant 10, by Miltenyi Biotec (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
4 protocols using entyvio
1
Immunohistochemical Analysis of Colonic Biopsies in IBD
2
Fluorescent Labeling of Therapeutic Antibodies
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Adalimumab (Humira 40 mg, Abbvie, 0,8mL solution for injection in pre-filled pen) and Vedolizumab (Entyvio 300 mg, Takeda, powder for concentrate for solution for infusion) were obtained from the Nantes University Hospital Pharmacy.
Before labelling procedure, Adalimumab and Vedolizumab were diluted to 2 mg/mL within 0.05M Na+ borate buffer. Antibodies labeling was performed according to APEX™ Antibody Labeling Kits protocol (Invitrogen™ #53027). This kit allowed to covalently attaching Alexa Fluor™ 647 to Adalimumab and FITC™ 488 to Vedolizumab. At the end of the procedure, presence of labelled antibodies was assessed by dot blot. Labelled-antibodies were stored at -80°C in aliquots of 20μg of proteins in PBS buffer with 0.1% NaN3.
Before labelling procedure, Adalimumab and Vedolizumab were diluted to 2 mg/mL within 0.05M Na+ borate buffer. Antibodies labeling was performed according to APEX™ Antibody Labeling Kits protocol (Invitrogen™ #53027). This kit allowed to covalently attaching Alexa Fluor™ 647 to Adalimumab and FITC™ 488 to Vedolizumab. At the end of the procedure, presence of labelled antibodies was assessed by dot blot. Labelled-antibodies were stored at -80°C in aliquots of 20μg of proteins in PBS buffer with 0.1% NaN3.
3
Comparative Analysis of Infliximab and Vedolizumab for UC
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Among biologics, only infliximab (Remicade®, intravenous injection, 100 mg/vial, Xian Janssen) and vedolizumab (Entyvio®, intravenous injection, 300 mg/vial, Takeda) have been approved for the treatment of UC in China. Hence, vedolizumab and infliximab were included for comparison in this study. Vedolizumab was given intravenously at weeks 0, 2, and 6 of the induction period and 300 mg once every 8 weeks in the maintenance period without dose escalation. Infliximab was injected 5 mg/kg at weeks 0, 2, and 6 of the induction period and every 8 weeks in the maintenance period without dose escalation.
4
Multiparametric Flow Cytometry for Immune Cell Profiling
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For flow cytometry, cells were stained according to standard protocols for 15 minutes at 4 °C using the following fluorochrome-conjugated extracellular antibodies: CD3 (VioGreen, REA613, Miltenyi Biotec), CD8 (PerCP/Cy5.5, RPA-T8, BioLegend; AF647, SK1, BioLegend), CD19 (VioBlue, Miltenyi Biotec), CD16 (APC/Cy7, 3G8, BioLegend), CD14 (AF488, HCD14, BioLegend, VioBlue, TÜK4, Miltenyi Biotec), CD56 (PE-Vio770, REA196, Miltenyi Biotec; FITC, HCD56, BioLegend), CCR3 (FITC, 5E8, BioLegend), Siglec 8 (PE-Dazzle594, 7C9, BioLegend), CD49d (VioBlue, MZ18-24A9, Miltenyi Biotec; PE/Cy7, 9F10, BioLegend), and integrin beta 7 (PE, FIB27, BioLegend; BV605, FIB504, BD Biosciences). Where indicated, VDZ (Entyvio, Takeda) was labeled using the Alexa Fluor Antibody Labeling Kits (AF674, Life Technologies) according to the manufacturer's instructions and used for staining. Viability staining was performed using Fixable Viability Dye eFluor 506 (Invitrogen) for 30 minutes at 4 °C.
Cells were fixed overnight using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) at 4 °C.
Data were acquired on LSR Fortessa (BD Biosciences), MACSQuant 10, and MACSQuant 16 (Miltenyi Biotec) instruments and analyzed with FlowJo single cell analysis software 7.6.5 and 10.06.1 (Tree Star Inc).
Cells were fixed overnight using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) at 4 °C.
Data were acquired on LSR Fortessa (BD Biosciences), MACSQuant 10, and MACSQuant 16 (Miltenyi Biotec) instruments and analyzed with FlowJo single cell analysis software 7.6.5 and 10.06.1 (Tree Star Inc).
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