For the extraction of the total protein, the cultivated cells were lysed using the lysis buffer [50 mM Tris (pH 7.4), 1% Triton X-100, 1% sodium 125 deoxycholate, 0.1% SDS, 150 mM NaCl, 1.0 mM EDTA, 1.0 mM Na3VO4, and inhibitor cocktail tablets]. A designated equal amount of the total proteins extracted from different treatments was resolved on a sodium dodecyl sulfate-polyacrylamide gel (i.e., 7.5%, 12.5% polyacrylamide gels) electrophoresis (SDS-PAGE) and transferred onto the nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were incubated with the primary rabbit monoclonal anti-HIF-1α from Life Technology Invitrogen (Thermo Fisher Scientific Corp., Waltham, MA, USA). Then, they were overlaid with the secondary polyclonal goat anti-rabbit antibodies (Abcam, Cambridge, UK). The protein bands were visualized using a chemiluminescence system (Millipore, Burlington, MA, USA).
Goat anti rabbit antibodies
Manufactured by Abcam
Sourced in China
Goat anti-rabbit antibodies are secondary antibodies used to detect and bind to rabbit primary antibodies in various immunological assays and techniques. These antibodies are produced by immunizing goats with rabbit immunoglobulins and then purifying the resulting antibodies.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Chemiluminescence system, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
β actin antibody, by Abcam (1 mentions)
β catenin, by Abcam (1 mentions)
Pvdf membrane, by Abcam (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Anti hsp47, by Abcam (1 mentions)
Anti α sma, by Abcam (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
5 protocols using goat anti rabbit antibodies
1
Extraction and Detection of HIF-1α Protein
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For the extraction of the total protein, the cultivated cells were lysed using the lysis buffer [50 mM Tris (pH 7.4), 1% Triton X-100, 1% sodium 125 deoxycholate, 0.1% SDS, 150 mM NaCl, 1.0 mM EDTA, 1.0 mM Na3VO4, and inhibitor cocktail tablets]. A designated equal amount of the total proteins extracted from different treatments was resolved on a sodium dodecyl sulfate-polyacrylamide gel (i.e., 7.5%, 12.5% polyacrylamide gels) electrophoresis (SDS-PAGE) and transferred onto the nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were incubated with the primary rabbit monoclonal anti-HIF-1α from Life Technology Invitrogen (Thermo Fisher Scientific Corp., Waltham, MA, USA). Then, they were overlaid with the secondary polyclonal goat anti-rabbit antibodies (Abcam, Cambridge, UK). The protein bands were visualized using a chemiluminescence system (Millipore, Burlington, MA, USA).
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For the extraction of the total protein, the cultivated cells were lysed using the lysis buffer [50 mM Tris (pH 7.4), 1% Triton X-100, 1% sodium 125 deoxycholate, 0.1% SDS, 150 mM NaCl, 1.0 mM EDTA, 1.0 mM Na3VO4, and inhibitor cocktail tablets]. A designated equal amount of the total proteins extracted from different treatments was resolved on a sodium dodecyl sulfate-polyacrylamide gel (i.e., 7.5%, 12.5% polyacrylamide gels) electrophoresis (SDS-PAGE) and transferred onto the nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were incubated with the primary rabbit monoclonal anti-HIF-1α from Life Technology Invitrogen (Thermo Fisher Scientific Corp., Waltham, MA, USA). Then, they were overlaid with the secondary polyclonal goat anti-rabbit antibodies (Abcam, Cambridge, UK). The protein bands were visualized using a chemiluminescence system (Millipore, Burlington, MA, USA).
2
Protein Expression Analysis in Traumatic Brain Injury
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On the 14th postoperative day, brain sections (6 × 4 × 3 mm) containing the centre of the impact point were obtained from the SD rats in each group (N = 5). RIPA lysis buffer was used to extract protein from the brain. Protein samples were electrophoresed using 10% SDS-PAGE and transferred to PVDF membranes (Abcam). Nonspecific reactivity was blocked with 5% skimmed milk in Tris-buffered saline containing Tween-20 for 60 min at room temperature. Membranes were incubated with rabbit anti-rat NSE, caspase-3, SOD, Wnt 3a (Abcam), and β-catenin (Abcam) antibodies at 4°C overnight, followed by incubation with goat anti-rabbit antibodies (Abcam). β-Actin antibodies (Abcam) were used as an internal control. The relative amounts of proteins were measured using Image Lab 8.0 (Media Cybernetics, USA).
3
Immunohistochemical Analysis of Skin and Vein Samples
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Histological sections (5 μm thick) were cut from te paraffin-embedded skin and vein samples and used for detection of different stromal cell types using specific fluorescent markers, as follows: FITC-labelled avidin (1:400) for mast cells [29 (link)]; FITC-labelled UEA lectin (1:10) for blood vessels [30 (link)]; rabbit polyclonal anti-HSP47 (1:50 Abcam, Milan, Italy) followed by FITC-labelled goat anti-rabbit antibodies (1:32 Abcam) for activated fibroblasts [31 (link)]; and goat polyclonal anti-α-SMA (1:400 Abcam) followed by FITC-labelled rabbit anti-goat antibodies (1:175 Abcam) for both blood vessels and myofibroblasts [32 (link)]. In some slides, nuclei were counterstained in red with propidium iodide. Before each immunolabeling, antigen retrieval was performed using 0.1 M citrate buffer at 96 °C for 10 min. The fluorescent markers and the primary antisera were applied overnight at 4 °C, and the secondary antisera for 2 h at 37 °C. Omission of the primary antibody was used as a negative control for the immunofluorescence reactions. The labelled sections were viewed and photographed using a Zeiss Axioskop UV microscope equipped with a digital camera and Axiovision 4 software (Zeiss, Jena, Germany) or a Leica TCS SP5 confocal microscope. Unless otherwise stated, all reagents were from Sigma-Aldrich.
4
Immunohistochemical Analysis of Cellular Proliferation
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The sections were sectioned and treated with xylene and a reduced ethanol gradient, followed by slow rinsing with tap water until the sections were clean and clear. After antigen repair with high pressure, the tissues were blocked with goat serum for 30 min. After incubation with the rabbit Ki67 antibody (Affinity, China), the blocked tissues were rinsed three times with PBS and then incubated with the goat anti‐rabbit antibodies (1:300, Abcam, China). Finally, the sections were subjected to DAPI (Beyotime, China) re‐staining, and the sections were scanned by an automated digital slide scanner. Proliferation was analyzed by calculating the percentage of positive follicles in the five corresponding microscopic fields.
5
Quantifying Mitochondrial ATP Expression
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In order to evaluate the function of mitochondria, the expression of ATP in tissues was observed using immunofluorescence technique after thawing. The tissue sections were hyalinized and then subjected to antigenic high‐pressure repair. The sections were then closed with goat serum. Rabbit anti‐ATP citrate lyase antibodies (1:200, Abcam, China) were incubated at 37°C for 1 h, followed by goat anti‐rabbit antibodies (1:300, Abcam, China) at 37°C for 30 min under light. Sections were then thoroughly cleaned with PBS before the nuclei were stained with DAPI (Beyotime, China) solution. The percentage of ATP expression was calculated using ImageJ software and a light microscope to examine the sections.
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