- 740021M20UG - Invitrogen Antibodies IL17A antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of IL-17A was performed using 70% confluent Neuro-2A cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were stained with IL-17A Recombinant Mouse Monoclonal Antibody (eBio64DEC17) (Product # 740021M, 1:100) at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor™ Plus 488 (Product # A32723) at a dilution of 1:2000 for 1 hour at room temperature. Panels a) shows representative image of cells that were stained for detection and localization of IL-17A (green). Panel b) shows representative image of cells stained for nuclei, using DAPI (Product # D1306, 1:500). Panel c) represents a composite image of panels a and b, clearly demonstrating cytoplasmic localization of IL17A. Panel d) represents no primary antibody control to assess the background. The images were captured on EVOS™ M7000 Imaging System (Product # AMF7000) at 20X magnification and externally deconvoluted.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of IL-17A was performed using formalin-fixed paraffin-embedded human tonsil tissue sections. To expose the target protein, heat-induced epitope retrieval was performed on de-paraffinized sections using eBioscience™ IHC Antigen Retrieval Solution - High pH (10X) (Product # 00-4956-58) diluted to 1X solution in water in a decloaking chamber at 110 degree Celsius for 15 minutes. Following antigen retrieval, the sections were blocked with 3% H2O2 for 1h at room temperature followed by 2% normal goat serum in 1X PBS for 45 minutes at room temperature and then probed with or without IL-17A Recombinant Mouse Monoclonal Antibody (eBio64DEC17) (Product # 740021M) at 1:500 dilution in 0.1% normal goat serum overnight at 4 degree Celsius in a humidified chamber. Detection was performed using Alexa Fluor™ 647 Tyramide SuperBoost™ Kit, goat anti-mouse IgG (Product # B40916). Nuclei were stained with DAPI (Product # D1306) and the sections were mounted using ProLong™ Glass Antifade Mountant (Product # P36984). The images were captured on EVOS™ M7000 Imaging System (Product # AMF7000) at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: HEK-293E cells were transiently transfected with human IL-17A construct (pink histogram) or left untransfected (blue histogram). Cells were fixed and permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (Product # 00-5523-00) and then stained intracellularly with 0.25 µg/test of IL-17A Recombinant Mouse Monoclonal Antibody (eBio64DEC17) (Product # 740021M) (pink histogram) or left untransfected (blue histogram), followed by Goat anti-Mouse IgG (H+L) Alexa Fluor™ Plus 488 (Product # A55058). Viable cells were used for analysis as determined by Fixable Viability Dye eFluor™ 780 (Product # 65-0865-14). The flow cytometry data was acquired using Attune™ NxT Flow Cytometer (Product # A29004).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of IL-17A was performed using EL4 cells and EL4 cells treated with PMA (50ng/ml) and Ionomycin (500ng/ml) for 6 hours, then with Brefeldin A (500ng/ml) for 18 hours. The cells were fixed and permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (Product # 00-5523-00) and then stained intracellularly with 2 µg/test of IL-17A Recombinant Mouse Monoclonal Antibody (eBio64DEC17) (Product # 740021M), followed by Goat anti-Mouse IgG (H+L), Alexa Fluor™ Plus 488 (Product # A55058). An increased expression of IL-17A was observed in treated cells (pink histogram) compared to untreated cells (blue histogram). Viable cells were used for analysis, as determined by LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit (Product # L34964). The flow cytometry data was acquired using Attune™ NxT Flow Cytometer (Product # A29004).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: A549 cells were fixed and permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (Product # 00-5523-00) and then stained intracellularly with 0.5 µg/test of IL-17A Recombinant Mouse Monoclonal Antibody (eBio64DEC17) (Product # 740021M) (pink histogram) or 0.5 µg/test of Mouse IgG1 K Isotype Control (Product # 14-4714-85) (blue histogram), followed by Goat anti-Mouse IgG (H+L) Alexa Fluor™ Plus 647 (Product # A55060). Viable cells were used for analysis, as determined by LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit (Product # L34964). The flow cytometry data was acquired using Attune™ NxT Flow Cytometer (Product # A29004).

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