SK-OV-3 cells were cultured in dishes as a 2D monolayer and in single layer biocomposites and exposed to normoxia (21% pO2) or to hypoxia (0.2% pO2) for 4h prior to fixation in 4% PFA for 10 minutes. TRACERS were cultured for 4h, unrolled and fixed in 4% PFA for 10 minutes. Immunostaning for HIF1α was conducted by first permeabilizing samples for 1h with 2% trition-X followed by washing with PBS, blocking with 5% normal goat serum, and application of rabbit anti-HIF1α (Cat # GTX61608, GeneTex) overnight. Secondary goat anti-rabbit TRITC was applied for 1h. Detection was conducted with a Zeiss LSM 700 by capturing 5um slices and assessing the HIF1α staining intensity in the nuclear regions (identified with the nuclear stain DRAQ5). Images were processed using FIJI.
Rabbit anti hif 1α
Manufactured by GeneTex
Sourced in United States
Rabbit anti-HIF-1α is a primary antibody that specifically recognizes the Hypoxia Inducible Factor-1 alpha (HIF-1α) protein. HIF-1α is a transcription factor that plays a central role in the cellular response to hypoxia. This antibody can be used in various immunological techniques to detect and study the expression and localization of HIF-1α.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti β actin, by GeneTex (2 mentions)
Mouse anti lamin a c, by BD (2 mentions)
Mouse anti β catenin, by BD (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Anti gapdh antibody, by Merck Group (1 mentions)
Ecl detection reagent, by Merck Group (1 mentions)
Rabbit anti α tubulin, by GeneTex (1 mentions)
Horseradish peroxidase conjugated rabbit anti mouse or goat anti rabbit secondary antibodies, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 protocols using rabbit anti hif 1α
1
Hypoxia Induces HIF1α Expression
2
Antibody Characterization for TMEM207
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Rabbit anti‐WWOX and anti‐GAPDH antibodies were obtained from Sigma‐Aldrich (St. Louis, MO, USA), while the rabbit anti‐HIF‐1α and anti‐GLUT‐1 were purchased from GeneTex (Irvine, CA, USA) and Spring Bioscience (Pleasanton, CA, USA), respectively. In this study, we mainly used a monoclonal antibody recognizing the synthetic peptide VNYNDQHPNGW (a.a. 40–50 of TMEM207), whereas an affinity‐purified rabbit antibody against human TMEM207 was applied for confirmation. The detailed procedure for the preparation and characterization of the two anti‐TMEM207 antibodies was described previously 14 , 15 .
3
Western Blot Analysis of Key Cellular Proteins
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Protein extraction was performed based on our previous report [14 (link)]. Primary antibodies were diluted 1:2000 and incubated overnight at 4 °C. Secondary antibodies diluted 1:5000 were added and incubated at room temperature for 1 h. Signals were detected using ECL detection reagent (Millipore Corporation, Burlington, MA, USA) following the manufacturer’s instructions. The primary antibodies used were as follows: mouse anti-β-catenin, mouse anti-lamin A/C (BD Biosciences, San Jose, CA, USA), mouse anti-c-myc, mouse anti-cyclin D1, rabbit anti-HDAC1, rabbit anti-HIF-1α and rabbit anti-β-actin (GeneTex, Irvine, CA, USA). Horseradish peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit secondary antibodies (GeneTex) were used as appropriate. Detailed information is given in the Supplementary Materials .
4
Western Blot Protein Detection Protocol
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Western blot assays were conducted as described previously [7 (link)]. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8% or 10% gels (Thermo) and were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). PVDF membranes were blocked in 1 mg/mL BSA (Sigma-Aldrich) at room temperature for 1 h. Primary antibodies were diluted 1:2000 and incubated overnight at 4 °C. Secondary antibodies diluted 1:5000 were added and incubated at room temperature for 1 h. The band intensities corresponding to the protein samples were measured using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The primary antibodies used were as follows: mouse anti-β-catenin, mouse anti-lamin A/C (BD Biosciences, CA, USA), mouse anti-TCF4 clone 6H5-3 (Millipore), mouse anti-N-EBP1 (C11) (Santa Cruz, CA, USA), rabbit anti-EBP1, rabbit anti-histone H3, rabbit anti-α-tubulin, rabbit anti-HIF-1α and rabbit anti-β-actin (GeneTex, CA, USA). Horseradish peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology) were used as appropriate.
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