A segment of sciatic nerves (1 cm) was fixed in 10% buffered formalin; 5-µm-thick transverse sections were repaired using antigen retrieval with hot citric acid buffer (pH 6.0) and blocked with 3% BSA. The sections were incubated with primary antibodies (Mfn1, 1:200, OM209507, omnimabs; Opa1, 1:200, ab157457, Abcam; Drp1, 1:200, ab184247, Abcam; MPZ, 1:50, ab183868, Abcam; S100, 1:100, ab4066, Abcam) at 4°C overnight followed by incubation with the secondary antibody and incubated at room temperature for 2 h. The sections were observed using Leica TCS SP8 STED and a Leica LAS image acquisition system. Image-Pro Plus 6.0 software was used for image analysis. Cells were plated in a 20-mm confocal dish and incubated with MitoTracker TM Deep Red FM and Drp1 antibody (1:250, ab184247, Abcam), as described in methods of 2.9.
Ab184247
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab184247 is an antibody product manufactured by Abcam. It is a polyclonal antibody raised in rabbit against a synthetic peptide. The antibody recognizes a specific protein target, but its core function is to bind to and detect the presence of that target protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab157457, by Abcam (9 mentions)
Ab124773, by Abcam (7 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Ab32503, by Abcam (5 mentions)
Ab13847, by Abcam (4 mentions)
Ab6721, by Abcam (4 mentions)
Fv1000, by Olympus (3 mentions)
Ab182858, by Abcam (3 mentions)
Ab2302, by Abcam (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Ab229969, by Abcam (3 mentions)
Ab8227, by Abcam (3 mentions)
Ab56889, by Abcam (3 mentions)
C1002, by Beyotime (2 mentions)
Ab189494, by Abcam (2 mentions)
Bs 10021r, by Bioss Antibodies (2 mentions)
Ab11306, by Abcam (2 mentions)
Ab131607, by Abcam (2 mentions)
Ab71498, by Abcam (2 mentions)
Sa00001 1, by Proteintech (2 mentions)
64 protocols using ab184247
1
Mitochondrial Dynamics in Sciatic Nerve Tissue
2
Immunohistochemical Analysis of Brain Tissue
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Brain tissues (n = 6) were put into 30% sucrose solution for 24 h and cut into 30 μm coronal sections using a Leica CM1950 cryostat (Leica Microsystems). After washing with PBS, coronal sections were sealed with 10% goat serum (C0265, Beyotime) and then incubated with primary antibodies of Drp1 (ab184247, Abcam) and Parkin (JF82-09, Novus). The sections were then sealed with 10% goat serum for 1 h and then incubated with Tomm20 antibody (H00009804-M01, Abnova). Thereafter, the sections were stained with fluorochrome-conjugated secondary antibody (ZF-0311, ZSGB-BIO), glial fibrillary acidic protein antibody (1 : 5000; ab7260, Abcam), and ionized calcium-binding adapter molecule 1 antibody (1 : 500; 019-19741, Wako). In brief, paraffin sections were immunostained with anti-nuclei (NeuN) antibody (ab177487, Abcam) at 4°C overnight and subsequently subjected to terminal deoxynucleotidyl transferase dUTP nick-end labeling staining using an In Situ Cell Death Detection kit (Cat. No. 11 684 795 910) according to the manufacturer's protocol. Finally, the sections were incubated with 4′,6-diamidino-2-phenylindole (C1002, Beyotime) nuclear dye, and the sections were visualized using a confocal laser scanning microscope (FV1000, Olympus). Sections from forebrain, midbrain, and hindbrain regions were randomly selected, and the ischemic penumbra was counted for statistical analysis.
3
Corneal Protein Expression Analysis
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Total protein was prepared from each cornea using radioimmunoprecipitation assay (RIPA) buffer (Galen, Beijing, China) and quantified. Western blot analyses were performed as we described previously [24 (link)]. For each sample, the levels of proteins of interest were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primary antibodies included dynamin-related protein 1 (DRP1) antibody (ab184247, Abcam), superoxide dismutase 2 (SOD2) antibody (ab68155, Abcam), and GAPDH antibody (KC-5G5, Kangchen, Shanghai, China).
4
Mitochondrial Dynamics and Apoptosis Regulation in Liver Cells
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The liver tissue and HepG2 cells were lysed by RIPA lysis solution (Beyotime, China) to extract the protein. The protein concentration was detected by BCA reaction kit. After quantitative analysis, the total protein was denatured in this study. SDS-Page gel was used for electrophoresis, electrophoresis apparatus (Bio-RAD, USA) was adjusted to 120 V for electrophoresis, PVDF membrane (Millipore, USA) was used for membrane transfer, and skim milk (Sigma, USA) for blocking. Primary antibodies (Abcam, UK): Sirt1 (1:1000; ab189494), optic atrophy 1 (Opa1, 1:1000; ab157457), mitofusin 2 (Mfn2, 1:1000; ab124773), Drp1 (1:1000; ab184247), NRF1 (1:1000; ab34682), mitochondrial transcription factor A (TFAM, 1:1000; ab252432; Abcam; UK), Bcl-2 (1:2000; ab182858), cleaved-caspase 3 (1:500; ab2302), BCL2-associated X protein (Bax, 1:1000; ab32503), cleaved-caspase 9 (1:2000; ab32539) and GAPDH (1:2500; ab9485) were then added overnight to incubate. The next day, goat anti-rabbit antibody (1:2000; ab288151) was incubated for 1 h with slow shaking at 25 °C. The immunoreactive bands were visualized by intensive chemiluminescent reagent. The gray value was analyzed by ImageJ software.
5
Protein Expression Analysis in HPAECs
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Protein samples were extracted from HPAECs by using ice-cold lysis buffer and then centrifuged at 13,500 rpm for 15 min at 4 ℃. After centrifugation, the protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). Protein samples (30 µg) were fractionated on 12% SDS-PAGE gels, transferred onto nitrocellulose membranes, and subsequently blocked with 5% nonfat milk at room temperature for 1 h. The membranes were incubated with specific antibodies against NLRP3 (2 µg/mL, bs-10021R, Bioss, Beijing, China), Caspase-1 (1 µg/mL, 22915-1-AP, Proteintech, IL, USA), pro-Caspase-1 (1 µg/mL, ab179515, Abcam, MA, USA), IL-1β (1 µg/mL, 16806-1-AP, Proteintech, IL, USA), DRP1 (1 µg/mL, ab184247, Abcam, MA, USA), YTHDC1 (2 µg/mL, 14392-1-AP, Proteintech, IL, USA), FTO (2 µg/mL, bs-7056R, Bioss, Beijing, China). Bands were sequentially incubated with horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h and enhanced chemiluminescent reagent imaging.
6
Immunohistochemical Analysis of Renal Proteins
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The 4% formaldehyde-fixed renal tissue sections were deparaffinized in xylene and rehydrated through graded ethanol. After antigen recovery was performed using a pressure cooker, the endogenous peroxidase was blocked with 3% H2O2 for 30 min at room temperature. Then the slides were blocked with 10% goat serum and were respectively incubated with primary antibodies against nestin (1:200; Abcam, #ab11306, RRID: AB_1640723 and #ab22035, RRID: AB_446723) and nephrin (1:200; Santa, Dallas, TX, USA, #sc-377246), Mfn1 (1:200; Proteintech, #13798-1-AP, RRID: AB_2266318) and Drp1 (1:200; Abcam, #ab184247) overnight at 4 °C. Then the sections were incubated with polymer helper and polyperoxidase-anti-mouse/rabbit IgG at 37 °C, and finally stained with diaminobenzidine. Finally images were captured using an Olympus microscope (OLYMPUS, BX71, Tokyo, Japan). The average integrated optical density value was quantified to indicate the expression of protein by Image Pro Plus (Media Cybernetics).
7
Visualizing NRF1 and Drp1 in HepG2 Cells
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In this study, we used prewarmed PBS to flush HepG2 cells 3 times and 4% paraformaldehyde to immobilize the cells for 20 min. Then, we used PBS (containing 5% BSA) to wash HepG2 cells for 60 min and used primary antibodies against nuclear respiratory factor 1 (NRF1; 1:200; ab200976; Abcam; UK) and Drp1 (1:250; ab184247; Abcam; UK) to incubate for 12 h at 4 °C. Subsequently, we used PBS to flush it 3 times and used secondary antibody goat anti-rabbit IgG (1:200, Abcam; ab150077; Abcam; UK) to hatch for 1 h at indoor temperature, protected from light. Nuclei were dyed with DAPI. Finally, we used Nikon Eclipse 80i microscope to observ sealed slides, and using Image-Pro Plus 6.0 to analyze the fluorescence intensity.
8
Mitochondrial Protein Extraction and Analysis in Cerebral Infarction
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Total proteins of infarcted cortex were extracted using the protein extraction kit (BB-3101, Bestbio), and mitochondrial proteins of infarcted cortex were isolated using Tissue Mitochondria Isolation Kit (C3606, Beyotime). The concentration of proteins was measured using the bicinchoninic acid method, and loading buffer was added to obtain equal concentrations (3 μg/μL). Samples were subjected to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred on a 0.45 μm polyvinylidene difluoride membrane. After washing with tris-buffered saline with 0.1% Tween® 20, the membrane was sealed using a rapid sealing solution for 20 min and then incubated with primary antibodies at 4°C overnight, and the corresponding secondary antibodies (bs-0296G, Bioss) were incubated for 2 h at room temperature.
The following primary antibodies were used: LC3B (L7543, Sigma), p62 (18420-1-AP, Proteintech), mTOR (#2983, Cell Signaling Technology), p-mTOR (#5536, Cell Signaling Technology), IL-1β (16806-1-AP, Proteintech), IL-6 (#12153, Cell Signaling Technology), TNF-α (BS1857, Bioworld Technology) and β-actin (#3700, Cell Signaling Technology), COX IV (ab202554, Abcam), Drp1 (ab184247, Abcam), and Parkin (JF82-09, Novus).
The following primary antibodies were used: LC3B (L7543, Sigma), p62 (18420-1-AP, Proteintech), mTOR (#2983, Cell Signaling Technology), p-mTOR (#5536, Cell Signaling Technology), IL-1β (16806-1-AP, Proteintech), IL-6 (#12153, Cell Signaling Technology), TNF-α (BS1857, Bioworld Technology) and β-actin (#3700, Cell Signaling Technology), COX IV (ab202554, Abcam), Drp1 (ab184247, Abcam), and Parkin (JF82-09, Novus).
9
Immunohistochemical Analysis of Mitochondrial Fission
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The paraffin-embedded sections of mouse left lung tissues were blocked with 3% hydrogen peroxide for 15 min to inactivate endogenous peroxidases and then incubated overnight at 4°C with rabbit monoclonal anti-Drp-1 (ab184247, 1 : 1000; Abcam, USA) and rabbit polyclonal anti-Fis-1 (ab229969, 1 : 750; Abcam, USA). PBS instead of primary antibody was used as a negative control for immunofluorescence staining. The sections were washed and subsequently incubated with a secondary antibody (PV-0023-2, Bioss, China) for 1 h at room temperature. Then the sections were stained with DAB until the stain developed. Six fields of immunohistochemical images were randomly selected and semi-quantitative analysis of Drp-1 and Fis-1 was performed using the ImageJ software as previously described [34 (link)].
10
Hyperoxia-induced Drp1 Regulation in Rat Lungs
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Protein was extracted from frozen lung tissue samples from 36 rats in the control and hyperoxia groups on days 3, 7, and 14, and mixed with the loading buffer. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 100 V for 3 h and transferred to polyacrylamide difluoride membranes at 100 V for 50 min. Membranes were then blocked in 5% skimmed milk for 3 h at room temperature (20–25°C). Membranes were then incubated with rabbit monoclonal anti-Drp1 (ab184247; 1:500 dilution; Abcam, Cambridge, UK) or rabbit Phospho-Drp1 (Ser616) Antibody (#3455; 1:1,000 dilution; Cell Signaling Technology, Boston, USA) and gently shaken at 4°C overnight. The next day, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (1:5,000 dilution; Cell Signaling Technology, Boston, USA) for 2 h after being washed three times in Tris-buffered saline and tween-20, and developed using enhanced chemiluminescence reagents (Thermo Scientific Pierce; Thermo Fisher Scientific, Waltham, MA, USA). Densitometry values of each sample were calculated by Image Lab 5.0 software (Bio-Rad, Hercules, CA, USA) for all bands and standardized relative to β-actin.
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