Anti-HIF-1 alpha antibody [mgc3]

5 product images

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  Flow Cytometry - Anti-HIF-1 alpha antibody [mgc3] (ab16066)

  Overlay histogram showing HeLa cells stained with ab16066 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab16066, 2μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [mgc3] (ab16066)

  IHC image of ab16066 staining HIF-1 alpha in human colon formalin fixed paraffin embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with Tris/EDTA buffer (pH9, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab16066, 10μg/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
  *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

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  This image is courtesy of an anonymous customer review

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [mgc3] (ab16066)

  ab16066 staining HIF-1-alpha in Human small intestine (IBD) and tonsil tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde; antigen retrieval was by heat mediation with an EDTA buffer (pH 9.0). Samples were incubated with primary antibody (1/800 in diluent + background reducers) for 20 minutess at 25°C. An undiluted Goat polymer was used as the secondary antibody.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HIF-1 alpha antibody [mgc3] (ab16066)

  Immunohistochemistry was performed on normal biopsies of deparaffinized Human tonsil tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/20 with ab16066 (left) or without primary antibody (negative control - right) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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  Western blot - Anti-HIF-1 alpha antibody [mgc3] (ab16066)

  Antibody specificity was demonstrated by CRISPR-Cas9 mediated knockout of target protein. A loss of signal was observed for target protein in HIF-1 alpha KO cell line compared to control cell lines using ab16066.
  

  Uncharacterized bands were observed in HeLa Cas9 samples at ~40 kDa and 45 kDa.

  All lanes: Western blot - Anti-HIF-1 alpha antibody [mgc3] (ab16066) at 1/2000 dilution

  Lane 1: HeLa wild type (untreated) at 30 µg

  Lane 2: HeLa wild type treated with 150uM CoCl2 for 48 hrs at 30 µg

  Lane 3: HeLa Cas9 at 30 µg

  Lane 4: HeLa Cas9 treated with 150uM CoCl2 for 48 hrs at 30 µg

  Lane 5: HeLa HIF1A KO at 30 µg

  Lane 6: HeLa HIF1A KO treated with 150uM CoCl2 for 48 hrs at 30 µg

  Secondary

  All lanes: Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP at 1/4000 dilution

  Predicted band size: 92 kDa

  Observed band size: 90 kDa