MaxiSorp Nunc 96-well plates used for enzyme-linked immunosorbent assays (ELISAs) were from Thermo Fisher Scientific (Waltham, MA, USA). Superblock-TBS and horseradish peroxidase-conjugated secondary antibodies were from Pierce Chemical Co (Rockford, IL, USA). Primary monoclonal antibodies and other immunodetection reagents were purchased from Abcam (Cambridge, MA, USA), EMD Millipore (now Millipore/Sigma, Billerica, MA, USA), Santa Cruz Biotechnology (Dallas, TX, USA), Vector Laboratories (Burlingame, CA, USA), Invitrogen (Carlsbad, CA, USA), or Life Technologies/Thermo Fisher, Inc. (Waltham, MA, USA). Fine chemicals were purchased from CalBiochem (Carlsbad, CA, USA) or Sigma-Aldrich (St. Louis, MO, USA).
Primary monoclonal antibody
Manufactured by Abcam
Sourced in United States
Primary monoclonal antibodies are laboratory reagents used for the detection and identification of specific target proteins or antigens. They are produced from a single clone of B cells and bind to a specific epitope on the target molecule.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse 80i, by Nikon (2 mentions)
Licor gam irdye680 secondary antibodies, by LI COR (2 mentions)
Maxisorp 96 well plates nunc, by Thermo Fisher Scientific (1 mentions)
Tissue tek oct, by Sakura Finetek (1 mentions)
Tyramide signal amplification kit, by PerkinElmer (1 mentions)
Hrp labeled igg secondary antibody, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Hrp labelled goat anti rabbit secondary antibody, by Abcam (1 mentions)
Tissue ros assay kit, by Genmed Scientifics (1 mentions)
Bx51 fluorescent microscope, by Olympus (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Hrp labeled goat anti rabbit secondary antibody, by Abcam (1 mentions)
13 protocols using primary monoclonal antibody
1
ELISA Protocol with Immobilized Antibodies
2
Microglia Profiling in Ischemic Stroke
3
Immunocytochemical Analysis of Neural Markers
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The assay was performed as previously described20 (link). Briefly, fixed cells were incubated overnight at 4 °C with primary monoclonal antibodies against Nestin (1:400), MAP2 (1:250) and TH (1:200) (Abcam, USA). After induction period, hMSCs were washed five times with PBS and incubated with fluoro isothiocyanate (FITC) conjugated secondary antibodies (1:500 dilution) for 1 hour at room temperature. Finally, after washing five tmes with PBS, cells were counter stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the cell nuclei. Cells were washed thrice with PBS to remove excess DAPI stain. Stained cells were examined using a fluorescence microscope equipped with a digital camera (Nikon Eclipse 80i, Japan).
4
Quantifying Intestinal Protein Expression
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The intestinal tissue proteins were extracted in line with ELISA assay. After protein quantification, the proteins were separated by electrophoresis and transferred onto the PVDF membrane. Later, the PVDF membrane was blocked with 5% defatted milk powder for 2 h, incubated with the TBST-diluted primary monoclonal antibodies (dilution, 1:300-1:500; Abcam, MA, USA) at 4° C overnight. Later, the membrane was further incubated with the HRP-labeled IgG secondary antibody (Abcam, MA, USA). The IgG was diluted with TBST at the volumetric ratio of 1:2000 for subsequent experiment. After incubation, the protein bands were detected with chemiluminiscence, and optical density (OD) was analyzed with the Image Pro-Plus 6.0 software, with GAPDH as the endogenous reference. The results were expressed as the OD ratio of target protein to endogenous protein.
5
Immunofluorescence Characterization of Neural Cells
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The assay was performed as previously described [13 (link)]. Briefly, cells fixed with chilled methanol: acetone and were incubated overnight at 4 °C with primary monoclonal antibodies against MAP2 (1:250), Nestin (1:400), DAT (1:200) and TH (1:200) (Abcam, Cambridge, MI, USA). Treatment with fluoro isothiocyanate (FITC) and texas red (TR) conjugated secondary antibodies (1:500 dil) for 60 min at room temperature. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain and visualize the cell nuclei. Stained cells were examined using a fluorescence microscope equipped with a digital camera (Nikon Eclipse 80i, Tokyo, Japan).
6
Western Blot Analysis of TLR4 Pathway
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Cells were inoculated into the 6‐well plate and stimulated with LPS for 12 h. The collected cells were washed with PBS for two times, added with 1.0 ml of RIPA lysis buffer (Beyotime Biotechnology Co., Ltd.) and lysed on ice for 30 min. After protein quantification, protein sample was subjected to electrophoresis and transferred to the PVDF membrane. The membranes were blocked with 5% skimmed milk powder for 2 h, incubated with primary monoclonal antibodies (including TLR4, MD2, MyD88 and p‐P65) (Abcam, dilution 1:500) diluted in TBST. The blot was subsequently reacted with HRP‐labelled goat anti‐rabbit secondary antibody (Abcam, dilution 1:2000). After incubation, the membranes were visualized with ECL, followed by optical density analysis by Image‐Pro Plus 6.0 software. GAPDH was used as the internal control, and the results were shown as the comparison of the optical density value between the target protein and the internal control protein.
7
Mitochondrial Protein Complex Analysis
8
Mitochondrial Protein Complex Analysis
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Mitochondria (0.4 mg protein) were solubilized with digitonin (8 g/g protein) and separated by Blue Native/SDS PAGE as described by Wittig et al.48 (link). The solubilization buffer contained 50 mM NaCl, 50 mM imidazole, 2 mM aminohexanoic acid, and 1 mM EDTA (pH 7.0). Samples were spun at 100,000 x g for 15 min and solubilized proteins were collected from the supernatant. Coomassie blue G-250 was added and the proteins were separated by electrophoresis on a 4–13% gradient Blue Native gel. Gel strips were cut, soaked in 1% SDS for 15 min at 37 °C, and horizontally placed on top of a gel set for 10% SDS-PAGE (10% T, 3% C). After the second electrophoresis, proteins were transferred to a PVDF membrane for Western Blot analysis. PVDF membranes were incubated with primary monoclonal antibodies (1 μg/ml, MitoSciences Inc.) in Odyssey blocking buffer containing 0.01% Tween-20. Fluorescent LiCor GAM-IRDye680 secondary antibodies were used at a dilution of 1:15,000. Proteins were visualized and quantified by the LiCor scanner.
9
Mitochondrial OXPHOS Supercomplex Analysis
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Mitochondrial isolation was carried out as previously described75 . To analyze mitochondrial OXPHOS supercomplexes, Blue-Native polyacrylamide gel electrophoresis (BN-PAGE) was performed with mitochondrial fractions prepared from the skin samples as described previously76 (link). Protein expression of mitochondrial OXPHOS subunits in the skin samples was carried out following standard immunoblots. A premixed cocktail containing primary monoclonal antibodies (Mitosciences, Eugene, OR, USA) against subunits of OXPHOS complexes was used to detect OXPHOS supercomplexes in BN-PAGE analyses and protein expression of OXPHOS subunits in immunoblot analyses. Voltage-dependent anion channel (VDAC) or β-actin antibodies were used as loading controls.
10
Quantifying Lung α-SMA and TRPV1 Expression
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Six-µm-thick lung sections from each paraffin block were deparaffinized with xylene and rehydrated in ethanol. For immunohistochemical detection of α-smooth muscle actin (α-SMA) and TRPV1, the lung sections were incubated overnight at 4℃ with a primary monoclonal antibody against α-SMA (titer 1:25; Abcam, Cambridge, MA, USA) and TRPV1 (titer 1:100; Abcam), or mouse serum as a negative control instead of the primary antibody. Immunoreactivity was detected by sequential incubations of lung sections with a biotinylated secondary antibody, followed by peroxidase reagent and the 3,3′-diaminobenzidine (DAB) chromogen (Invitrogen, Carlsbad, CA, USA). The area in each paraffin-embedded lung immunostained by α-SMA was outlined and quantified using a light microscope attached to an image analysis system (BX50; Olympus, Tokyo, Japan). The results were expressed as the immunostained area of the bronchiolar basement membrane (internal diameter 150–200 µm). At least 10 bronchioles were counted in each slide.
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