Sections were stained with an H&E Staining Kit (KeyGen) and TRAP staining kit (Sigma-Aldrich). An alkaline phosphatase (ALP) staining kit (Jiancheng) was utilized to detect bone formation. For immunofluorescence staining, the primary antibodies used were rabbit anti-HIF-1α (1:200; Abcam) and mouse anti-CTSK (1:200; BBI). Then, the slides were incubated with DyLight 488-conjugated goat anti-mouse IgG (1:500; Abbkine) or DyLight 594-conjugated goat anti-rabbit IgG (1:500; Abbkine) followed by DAPI (1:800; Sigma-Aldrich).
Rabbit anti hif 1α
Manufactured by Abcam
Sourced in United States, United Kingdom
Rabbit anti-HIF-1α is a primary antibody that binds to the HIF-1α (Hypoxia-inducible factor 1-alpha) protein. HIF-1α is a subunit of the HIF-1 transcription factor, which plays a central role in the cellular response to hypoxia.
Automatically generated - may contain errors
Lab products found in correlation
Secondary rabbit envision, by Agilent Technologies (2 mentions)
Horse serum, by Biocare Medical (2 mentions)
Decloaker, by Biocare Medical (2 mentions)
Mec13, by BioLegend (2 mentions)
Citrate buffer ph 6, by Biocare Medical (2 mentions)
67 antibody d2h10, by Cell Signaling Technology (2 mentions)
Mec14, by Novus Biologicals (2 mentions)
Rat on mouse hrp polymer and probe, by Biocare Medical (2 mentions)
Dylight 488 conjugated goat anti mouse igg, by Abbkine (1 mentions)
Dylight 594 conjugated goat anti rabbit igg, by Abbkine (1 mentions)
Trap staining kit, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
H e staining kit, by Keygen Biotech (1 mentions)
Alp staining kit, by Nanjing Jiancheng (1 mentions)
Sds page, by Beyotime (1 mentions)
Anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti mouse igg, by Cell Signaling Technology (1 mentions)
Rabbit anti hk2, by Cell Signaling Technology (1 mentions)
Rabbit anti sirt6, by Abcam (1 mentions)
Mouse anti β actin, by ZSGB-BIO (1 mentions)
13 protocols using rabbit anti hif 1α
1
Histological Analysis of Bone Formation
2
Western Blot Analysis of SIRT6, HIF-1α, and HK2
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The cells were washed three times with PBS, and protein lysate buffer containing 5 µL PMSF (1 mM) per 500 µL was placed on ice for 5 min and centrifuged at 14,000×g for 10 min after sonication. Then, the supernatant was collected. The protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). After boiling with loading buffer for 10 min, equal amounts of protein (40 µg) were separated by 10% SDS–PAGE (Beyotime, Shanghai, China) and transferred onto PVDF membranes (Millipore, Massachusetts, USA). Membranes were blocked with 5% milk powder for 60 min at room temperature and incubated with specific primary antibodies at 4 °C overnight. Rabbit anti-SIRT6 (1:1000 dilution, Abcam, UK), rabbit anti-HIF-1α (1:500 dilution, Abcam, UK), rabbit anti-HK2 (1:500 dilution, Cell Signaling Technology, USA), and mouse anti-β-actin (1:1000 dilution, ZSGB-Bio, Beijing, China) antibodies were used. Proteins were detected using anti-rabbit IgG (1:2000 dilution, Cell Signaling Technology, USA) or anti-mouse IgG (1:2000 dilution, Cell Signaling Technology, USA) antibodies and a chemiluminescence detection system (FluorChem HD2, USA).
3
Western Blot Analysis of Protein Expression
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Each protein extract was adjusted to a 2 μg/μL concentration with the addition of Laemmli sample buffer (4X concentrate, Bio-Rad, Hercules, CA, USA), heated for 5 min at 95 °C, and separated on precast polyacrylamide gradient gels (7.5–15%). After transfer to 0.2 μm polyvinylidene difluoride membranes (PVDF, Bio-Rad), the membranes were blocked in either 5% (w/v) nonfat dried milk or 1% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich, Saint Louis, MO, USA) diluted in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBS-T), and probed overnight at 4 °C with the respective primary antibodies, as follows: mouse anti-actin (1:2500), rabbit anti-LDHA (1:1000), rabbit anti-microtubule-associated protein 1B/2B light chains 3B (MAP-LC3) (1:1000) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-HIF-1α (1:250), rabbit anti-PDK1 (1:1000), rabbit anti-Bax (1:1000), rabbit anti-Bcl-2 (1:500), and rabbit anti-caspase 3 (1:250) (Abcam, Cambridge, UK). Membranes were subsequently incubated with the respective HRP-linked anti-rabbit or anti-mouse IgG (1:1000) (Cell Signaling Technology, Danvers, MA, USA) antibodies and developed using ECL or ECL Plus (WesternBright Sirius Chemiluminescent Detection Kit, Advansta Inc., USA). Detection and densitometric quantification of band intensities was performed using a G-Box system (Syngene, Cambridge, UK).
4
Immunohistochemical Analysis of Tumor Samples
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Tumor sections were preserved in OCT compound, frozen and sectioned. 5 μm thick sections were fixed in 35/65 methanol/acetone. CD31 was detected with anti-mouse CD31 antibody (MEC13.3, Biolegend, San Diego CA). For the remaining IHC staining, Formalin-fixed tissue samples were paraffinized and sectioned by University of Iowa Comparative Pathology Core. Sections were then de-paraffinized in a series of xylene washes and rehydrated with ethanol and water. Antigen retrieval was accomplished by immersing slides in Citrate Buffer pH 6.0 in a decloaker (Biocare Medical, Concord CA). Tissue samples were incubated with 3% hydrogen peroxide followed by blocking with horse serum (Biocare Medical). Slides were then incubated with rabbit anti-mouse Ki-67 antibody (D2H10, Cell Signaling Technology), rat anti-CD34 antibody (MEC14.7, Novus Biologicals, Littleton CO) or rabbit anti-HIF1α (polyclonal, Abcam, Cambridge UK). Slides were then stained with Rat-on-mouse HRP polymer and probe (Biocare,) or secondary rabbit envision (DAKO, Santa Clara CA). The slides were developed with 3,3’-diaminobenzadine (DAB and 0.3% H2O2 in PBS) developing buffer, followed by counterstaining with hematoxylin. Positive staining was quantified using Image J software. All analysis of IHC was done blinded in regards to group allocation.
5
Western Blot Analysis of HIF-1α
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Forty (40) μg of total protein was resolved using 12% Tris-Tricine SDS-PAGE and Western blot was carried out as described by Sepramaniam et al [26 (link)]. Membranes were probed with primary antibody (rabbit anti HIF-1α, Abcam, UK) at a concentration of 1: 5,000 in 0.5% blocking solution. β-actin was used as a loading control [26 (link),30 (link)].
Secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit, Bio-Rad Laboratories Inc, PA, USA) was used at a dilution of 1:10,000 in 0.5% blocking solution. The membranes were washed and visualized using the enhanced chemiluminescence reagents (SuperSignal West; Thermo Scientific, USA) with variable exposures on Kodak-MS film. Films of Western blots were scanned (Acer SWZ3300U) and intensities of the corresponding bands were quantitated using Image J software (National Institutes of Health, USA).
Secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit, Bio-Rad Laboratories Inc, PA, USA) was used at a dilution of 1:10,000 in 0.5% blocking solution. The membranes were washed and visualized using the enhanced chemiluminescence reagents (SuperSignal West; Thermo Scientific, USA) with variable exposures on Kodak-MS film. Films of Western blots were scanned (Acer SWZ3300U) and intensities of the corresponding bands were quantitated using Image J software (National Institutes of Health, USA).
6
Immunofluorescence and Immunohistochemistry Protocols
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Cells or tissue sections were fixed in 4% paraformaldehyde for 15 min, washed in PBS, and permeabilized in 0.3% Triton-X in PBS for 10 min. Samples were washed thrice with PBS and then blocked with 5% donkey serum in PBS. Samples were then incubated with primary antibodies at 1:50–1:200 dilution at 4°C overnight and then incubated with Alexa-conjugated secondary antibodies for 1 h at room temperature. A Zeiss LSM 900 laser scanning confocal microscope was used to visualize the sections. For IHC analysis, paraffin sections were dewaxed with xylene and gradient ethanol. The sections were antigen-repaired with citric acid (pH 6.0) and blocked with Bovine Serum Albumin (BSA). The sections were incubated with rabbit anti- HIF1α (1:200, Abcam, USA) or rabbit anti- phospho-CaMKII (1:200, CST, USA) at 4°C overnight. Then the sections were incubated with goat anti-rabbit IgG-HRP secondary antibody (1:200, Bioss, China) at room temperature for 60 min. Visualized staining of tissue was performed with a fluorescence microscope.
7
Western Blotting of HIF-1α in RAW264.7-asc Cells
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Western blotting was performed as previously described [14 (link)]. Briefly, RAW264.7-asc cells were lysed in RIPA lysis buffer and boiled for 10 min. After centrifugation, 25 μg of total protein extract was separated by 12% SDS-PAGE, followed by transferring to PVDF membranes for Western blotting. After blocking with 5% milk dissolved in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBST) for 1 h at room temperature, the membranes were incubated with indicated with rabbit anti-HIF-1α (Abcam). Then mouse secondary antibody (Xiamen Bosheng Corp.) conjugated with horseradish peroxidase was used to detect the signal. Positive band intensities were detected using a gel documentation system (LAS-3000 Fujifilm Medical Systems, Stamford, CT).
8
Immunohistochemical Analysis of Tumor Samples
9
Immunohistochemical Analysis of Cell Markers
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The tissue sections were dewaxed and debenzylated according to previous study [37 (link)]. and then incubated with rabbit anti-Ki67 (Abcam, USA), rabbit anti-HIF-1α (Abcam, USA), rabbit anti-NCR1 (Abcam, USA) and rabbit anti-CD3 (CST, USA) antibodies overnight at 4 °C. After washing, the bound antibodies were detected using horseradish peroxidase-conjugated secondary antibody (DAKO, Denmark) and diaminobenzidine (DAKO, Denmark), followed by counterstaining with hematoxylin (Keygen Biotech, Nanjing, China). The images were viewed under a fluorescence microscope (LEICA DMI 4000B, Solms, Germany) and were analyzed by Leica application suite software (version 4.0).
10
Multimodal Tumor Therapy Comparison
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4T1 tumor-bearing mice (n = 3) were randomly assigned to four groups and treated with (a) AZGL (No light), (b) AZGL (PDT), (c) AZGL (PTT), (d) AZGL (PDT + PTT). Briefly, 200 µL of AZGL solution (1 mg/mL) were injected into the mice by the intravenous method, 14 days post-treatment, the collected tumor tissues were fixed and sliced. The tumors were excised and fixed in 4% formalin for HIF-1α staining. The slices were stained with rabbit anti-HIF-1α (1:1000, Abcam) monoclonal antibody overnight at 4 °C. Subsequently, the secondary antibodies conjugated with FITC were incubated with slices for 1 h. After staining with DAPI, the immunofluorescence staining images were captured using CLSM.
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