Under anesthesia, P10 pups were transcardially perfused with ice-cold PBS (pH 7.4) followed by infusion of 4% paraformaldehyde. Brains were then removed and fixed in 4% paraformaldehyde at 4°C for a minimum of 3 days. After dehydrated with 30% sucrose in PBS (pH 7.4), brains were processed to obtain 10-µm tissue slides. As described previously (Li et al., 2012b (link)), immunofluorescence was performed using the following primary antibodies: mouse anti-5mC (1:500; Active Motif, Carlsbad, CA), rabbit anti-HIF-1α (1:50; Santa Cruz) and mouse anti-neuronal nuclei (1:100; Millipore). After blocking with 1% bovine serum albumin in PBS containing 0.5% Triton X-100 for 2 h at room temperature and incubation with the primary antibodies at 4°C overnight, tissue sections were treated with secondary antibodies raised against mouse and rabbit IgG conjugated with Alexa Fluor 488 (1:200) and Alexa Fluor 647 (1:200) (Invitrogen, Carlsbad, CA), respectively, for 2 h at room temperature. After 3 washes, sections were stained with Hoechst 33258 (5 µg/mL; Sigma) for 1 min. The sections were then covered with aqueous mounting medium (RD Systems, Minneapolis, MN), visualized and imaged using the Zeiss LSM 710 confocal microscope, as previously described (Li et al., 2012b (link)).
Rabbit anti hif 1α
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Rabbit anti-HIF-1α is a polyclonal antibody that recognizes the hypoxia-inducible factor-1 alpha (HIF-1α) 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
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Mouse anti 5mc, by Active Motif (1 mentions)
Mouse anti neuronal nuclei, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Microtome, by Leica (1 mentions)
Xylazine, by Merck Group (1 mentions)
Rabbit anti ps6, by Cell Signaling Technology (1 mentions)
Signalstain boost immunohistochemistry detection reagent, by Cell Signaling Technology (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Takara kit, by Takara Bio (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Rabbit anti creb, by Abcam (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Anti gap43, by Abcam (1 mentions)
Rabbit anti ang 1, by Abcam (1 mentions)
Rabbit anti ang 2, by Abcam (1 mentions)
Rabbit anti parp, by Cell Signaling Technology (1 mentions)
Rabbit anti glut1, by Abcam (1 mentions)
10 protocols using rabbit anti hif 1α
1
Immunofluorescence Analysis of Brain Tissue
2
Quantifying HIF1α and p-S6 in Rat Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rats were anesthetized using 250 mg/kg ketamine and 2% xylazine (10 mg/kg, Sigma-Aldrich) for a duration of 6, 12 or 24 h following surgery, and were subsequently sacrificed by rapid decapitation. The collected brain tissues were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 8 h at 0°C. Fixed brains were paraffin embedded and cut into 2 µm sections using a microtome (Leica Microsystems GmbH, Wetzlar, Germany). Sections were briefly washed with TBS and incubated for 30 min in 3% H2O2 to quench endogenous peroxidase activity. The following primary antibodies: Rabbit anti-HIF1α (1:100; catalog no. sc-10790; Santa Cruz Biotechnology, Inc., Dallas; Texas; U.S.A) and rabbit anti-p-S6 (1:100; catalog no. 4858; Cell Signaling Technology, Inc.) were applied overnight at 4°C. Subsequently, sections were washed three times with TBS and then incubated with SignalStain® Boost Immunohistochemistry Detection reagent (1:50; catalog no. 8114; Cell Signaling Technology, Inc.) for 1 h at room temperature. Sections were then incubated with 3,3′-diaminobenzidine (ZSGB-BIO, Beijing, China). Finally, the sections were dehydrated, mounted and examined under a light microscope (Leica Microsystems GmbH). Positive cells were counted using Image-Pro Plus software verison 7.0 (Media Cybernetics, Inc., Rockville, MD, USA).
3
Hypoxia-Induced Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary Antibodies were rabbit anti-HIF-1α (Santa Cruz, USA). Acridine orange and rapamycin were obtained from Sigma and Invivogen, respectively. Quantitative RT-PCR using Takara Kit was purchased Takara (Japan). All other reagents were obtained from Sigma-Aldrich (Taufkirchen, Germany)
4
Western Blot Analysis of Brain Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At the same time points following the procedures [32 (link)], proteins were extracted from the MCA-supplied brain regions and loaded onto SDS-polyacrylamide gel for electrophoresis. Gel transfer to a PVDF membrane was performed under 200 V for 1 h. Membranes were blocked with 5% skimmed milk. Membranes were incubated with primary antibodies (1:1000, rabbit anti-BDNF, rabbit anti-NGF, rabbit anti-PSD-95, rabbit anti-SYN, mouse anti-Tau, rabbit anti-GAP43, rabbit anti-VEGF, rabbit anti-Ang-1, rabbit anti-Ang-2, rabbit anti-TrkB and rabbit anti-CREB, Abcam, MA, USA; 1:500, rabbit anti-HIF-1α, Santa Cruz Biotechnology, Inc. CA, USA) for 24 h at 4 °C. The secondary antibody was goat anti-rabbit IgG-HRP (Santa Cruz), which was incubated for 1 h at room temperature for all primary antibodies. Western blot images for each of the antibodies were analyzed using an image analysis program (ImageJ 1.42, National Institutes of Health, Bethesda, MD, USA) to quantify protein expression in terms of relative image density.
5
Western Blot Analysis of Cell Adhesion Proteins
6
Immunophenotyping of Neural Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neurospheres were allowed to adhere on poly-L-lysine coated glass coverslips (15µg/ml), fixed with 4% paraformaldehyde in PBS, for 10 min, at RT and incubated with following antibodies: monoclonal anti-nestin (1:100, Chemicon International, Temecula, CA, USA), anti-CD133/1 (1:50, Miltenyl Biotec. Inc., Auburn, CA, USA), anti-β-tubulin III (1:500, Promega, Mannheim, Germany), polyclonal anti-SOX2 (1:500, Abcam) and anti-GFAP (1:200, Sigma), rabbit anti-HIF-1α (1:200, Santa Cruz, CA, USA), anti-PPARɑ and anti-PPARγ (1:400, Thermo Scientific Inc., USA), anti-GLUT3 (1:100, Abcam, Cambridge, UK) and mouse IgM anti-glycogen (1:400, generous gift from Prof. Yoshinobu Baba, Nagoya University). Primary antibodies were revealed by Alexa Fluor 488 or 633 anti-rabbit IgG or Fluorescein Isothiocyanate (FITC) anti-mouse IgM secondary antibody (Sigma).
For bodipy staining, neurospheres were incubated with 4,4-difluoro-1,3,5,7,8-pentamethyl 4-bora-3a,4a-diaza-sindacene (1µg/mL, BODIPY 493/503 Molecular Probes, Invitrogen), for 10 min, at RT. Coverslips were mounted with Vectashield Mounting Medium with Dapi (Vector Laboratories, Burlingame, CA, USA) and examined at a Leica TCS SP5 confocal microscope (Mannheim, Germany).
For bodipy staining, neurospheres were incubated with 4,4-difluoro-1,3,5,7,8-pentamethyl 4-bora-3a,4a-diaza-sindacene (1µg/mL, BODIPY 493/503 Molecular Probes, Invitrogen), for 10 min, at RT. Coverslips were mounted with Vectashield Mounting Medium with Dapi (Vector Laboratories, Burlingame, CA, USA) and examined at a Leica TCS SP5 confocal microscope (Mannheim, Germany).
7
Immunoblotting Analysis of Protein Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell extracts were lysed in SDS buffer, a protease inhibitor cocktail (Promega) and a phosphatase inhibitor cocktail (FUJIFILM Wako Pure Chemical Corporation). After quantifying protein concentrations using BCA reagent (Thermo Fisher Scientific), proteins were resolved by SDS‐PAGE (polyacrylamide gel electrophoresis) and transferred to nitrocellulose membrane by electroblotting. Membranes were blocked in TBS containing 5% skim milk and probed with the following primary antibodies: goat anti‐galecint‐1 (1:1000, R&D systems), mouse anti‐TSC22D3 (1:500), mouse anti‐ubiquitin (1:500, Santa Cruz Biotechnology), rabbit anti‐HIF‐1α (1:1000), rabbit anti‐phosphorylated AKT (1:2000), rabbit anti‐AKT (1:2000), rabbit anti‐phosphorylated ERK1/2 (1:2000), rabbit anti‐ERK1/2 (1:2000, Cell signaling technology) and rabbit anti‐β‐actin (1:4000, Medical & Biological Laboratories) antibodies. Horseradish peroxidase‐conjugated anti‐goat, anti‐mouse and anti‐rabbit IgGs (Jackson ImmunoResearch Laboratories) were used as a secondary antibody for chemoluminescence detection. Signals were visualized using a SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).
8
Western Blot Analysis of PHD1 and HIF-1α
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primary antibodies used for the Western blot assay were mouse anti-β-actin (1:5000, abcam), rabbit anti-PHD1 (1:400, Novusbio), and rabbit anti-HIF-1α (1:500, Santa Cruz Biotech). Enhanced chemiluminescence (Thermo Fisher Scientific) was used for protein visualization. Three independent experiments were performed, and one representative result is shown. The expression level proteins were finally analyzed using Image Lab software densitometrically (details listed in the Supplemental Data).
9
Western Blot Analysis of Ischemic Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 3, 14, and 28 days after initiation of the exercise regimens, rats were sacrificed for Western blot analysis. Tissue samples from the ipsilesional ischemic cerebral hemispheres of all experimental groups were harvested, and total protein extraction was performed using cell lysis solutions (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Protein concentration was then determined by the BCA method. Electrophoresis (10% SDS-PAGE gel) was performed with 30 μg of protein per lane. Gel transfer to a PVDF membrane was performed under 200 V for 1 h. Membranes were blocked with 5% skimmed milk, followed by incubation with primary antibodies (1:1,000 rabbit anti-BDNF, rabbit anti-NGF, rabbit anti-PSD-95, rabbit anti-SYN, rabbit anti-Tau, and rabbit anti-GAP43, Abcam, MA, USA; 1:500 rabbit anti-HIF-1α, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. The next day, membranes were washed three times and further incubated with a goat anti-rabbit IgG-HRP secondary antibody (1:1,000, Santa Cruz) at room temperature for 1 h. After washing, the ECL method was used to detect signals. Western blot images for each antibody were analyzed using an image analysis program (ImageJ 1.42, National Institutes of Health, Bethesda, MD, USA) to quantify protein expression according to relative image density.
10
Western Blot Analysis of Cellular Fractions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell membrane fractions, whole cell lysate, or co-immunoprecipitated samples were resolved using 10% SDS-PAGE. For cell lysate and membrane fraction blots, 40 µg and 100 µg of protein was loaded, respectively. The separated proteins were then transblotted onto nitrocellulose membranes and the membranes were blocked using 2% casein in PBS + 0.05% Tween-20 (PBST). Blots were then probed overnight at 4 °C with monoclonal mouse anti-GAPDH antibody (1 µg/mL) (Calbiochem, San Diego, CA, USA), rabbit anti TfR2 (1 µg/mL) (Abcam, Cambridge, UK), or rabbit-anti HIF-1α (Santa Cruz Biotechnology, Dallas, TX, USA). Bound antibodies were then detected using anti-mouse peroxidase or anti-rabbit peroxidase antibodies (Sigma-Aldrich), respectively. After extensive washing with PBST, blots were developed using Luminata TM Forte Western HRP substrate (MilliporeSigma, Burlington, MA, USA). As a loading control, anti beta-actin was used for cell lysates. For membrane fractions, amido black staining was utilized to ensure equal protein loading. Blots were imaged on an ImageQuant LAS 4000 imager (GE Healthcare Life Sciences, Marlborough, MA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!