All cell lines were cultured on sterile glass slides. The cells were then fixed with Roti-Histofix 4% (Carl Roth, Karlsruhe, Germany) for 15 min, washed with PBS (Sigma/Merck, Darmstadt, Germany), blocked with 5% milk (Th. Geyer, Berlin, Germany) in PBS and permeabilized with 0.01% Triton X-100 (Carl Roth). Incubation with the anti-MUC1 mouse antibody (Cell Signaling) was performed overnight at 4 °C. Direct FITC-labeled anti-EpCAM (Acris) and anti-pan cytokeratin (CK8, CK18, CK19, Abcam) antibodies, as well as the secondary anti-mouse antibody (Dianova), were applied the next day for 1 h at RT. Cell nuclei were visualized using Hoechst 33258 (Sigma, Darmstadt, Germany). Images were taken using an inverted fluorescence microscope at 20× magnification (Carl Zeiss Microscopy).
Anti mouse secondary antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-mouse secondary antibody is a laboratory reagent used in immunoassays and other immunological techniques. Its core function is to bind to and detect primary antibodies raised against mouse antigens, enabling visualization or quantification of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Anti β actin antibody, by Abcam (4 mentions)
Anti rabbit secondary antibody, by Abcam (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Anti cd133, by Abcam (3 mentions)
Fluorchem fc2, by Bio-Techne (3 mentions)
Anti cd44, by Abcam (3 mentions)
Milli q purification system, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Anti gfp antibody, by Novus Biologicals (2 mentions)
Ecl reagent, by Merck Group (2 mentions)
Hrp conjugated anti rabbit, by Abcam (2 mentions)
Microscopy, by Zeiss (1 mentions)
Anti epcam, by OriGene (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Triton x 100, by Carl Roth (1 mentions)
Roti histofix 4, by Carl Roth (1 mentions)
Chemidoctm system, by Bio-Rad (1 mentions)
Primary antibody against caspase 3, by Cell Signaling Technology (1 mentions)
27 protocols using anti mouse secondary antibody
1
Immunofluorescence Staining of Cell Lines
2
Caspase-3 and Hsp70 Expression in U-937 Cells
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Cultured U-937wt cells were treated with BT44 at a concentration of 50 μM, then 2 μM of etoposide was administered 6 h later. After 15 h of incubation, cells were lysed in RIPA buffer, separated by PAGE on a 15% polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with PBS containing 5% (w/v) skimmed milk and incubated with a primary antibody against Caspase-3 (Cell Signaling, Danvers, MA, USA) and secondary antibodies (Abcam, Cambridge, UK) at room temperature for 1 h. Band intensity was quantified using the ChemiDocTM system (Bio-Rad, Hercules, CA, USA). To assess the content of Hsp70 in treated cells, 20 μg of the same cell lysates were separated by PAGE on an 11.5% gel. The membrane was probed with an anti-Hsp70 monoclonal antibody (Clone 2H9) followed by a secondary anti-mouse antibody (Abcam).
3
Quantification of HNE and Cathepsin G Activities
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Antibodies for detection of AAT and secondary anti-mouse antibodies were obtained from Abcam (Cambridge, UK). HNE, together with a bicinchoninic acid (BCA) protein assay kit were obtained from Thermo Scientific (Rockford, IL, USA). The standard p-nitroaniline (p-NA) and the peptide substrates used for the determination of human HNE and Cat. G activities (MeOSuc-Ala-Ala-Pro-Phe-NA and Suc-Ala-Ala-Pro-Phe-NA, respectively) were from Bachem (Bachem AG, Bubendorf, Switzerland). Unless otherwise stated, all other analytical grade reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Double-distilled water used for the preparation of all buffers was prepared with a Millipore (Bedford, MA, USA) Milli-Q purification system.
4
Immunological Detection of Proteins
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Antibodies for detection of AAT, HNE, and secondary anti-mouse antibodies were obtained from Abcam (Cambridge, UK) and Thermo Scientific (Rockford, IL, USA). Bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Scientific (Rockford, IL, USA). Unless otherwise stated, all other analytical grade reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Double-distilled water used for the preparation of all buffers was prepared with a Millipore (Bedford, MA, USA) Milli-Q purification system.
5
Quantitative Analysis of Yeast Ubiquitination
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Western blot analysis of yeast lysates was performed to assess the accumulation of polyubiquitinated proteins as described earlier [19 (link)]. Western blot analysis of the Rad52-3ha levels was performed in the same way. The pRad52-3ha plasmid-expressing Rad52, fused with the C-terminal 3xHA epitope from its native promoter, was assembled from PCR fragments by recombinational cloning [79 (link)]. PCR fragments used in the plasmid assembly were obtained using the primers listed in Table S2 . The Rad52-3ha levels were determined using a primary mouse monoclonal anti-HA antibody (1:1000, Sigma, USA) and an anti-mouse secondary antibody (1:100,000, Abcam, Cambridge HQ, UK). Tubulin was used as a loading control and was detected using a primary rat monoclonal antibody (1:1000, Abcam, UK) and an anti-rat secondary antibody (1:100,000, Abcam, UK). The images obtained were analyzed using ImageJ (https://imagej.nih.gov/ij/ ). The protein intensities were normalized to the actin or tubulin intensity.
6
Quantifying NGF Receptor Expression
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Twenty-four hours in advance, ISCs were plated in a 6-well plate at a density of 1×106 cells/mL and were treated with the different concentrations of mNGF described above. Proteins were extracted using a lysis buffer (radio immunoprecipitation assay : 1% triton X-100, 1% deoxycholate, 0.1% SDS; 1 mM PMSF). The total protein was measured using a BCA Protein Assay Kit (Beyotime). Samples containing equal amounts of protein (10 µg) were run on a 10% SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, Billerica, MA, USA) in Tris-glycine transfer buffer (Beyotime) at 300 mA for 90 minutes. The membranes were blocked with 5% nonfat milk (Bio-Rad), incubated with a primary antibody (rabbit monoclonal anti-TrkA antibody (Abcam, cat. no. ab76291; Cambridge, MA, USA) and rabbit monoclonal antip75NTR antibody (Abcam, cat. no. ab52987) in 5% BSA in tris buffered saline (TBS) overnight at 4°C, washed in TBS containing 0.05% (v/v) Tween 20, and incubated for 1 hour at room temperature with an HRP-conjugated anti-rabbit (Abcam) or anti-mouse secondary antibody (Abcam). The blots were developed using enhanced chemiluminescence (Beyotime) and ChemiDoc™ XRS+ System with Image Lab™ Software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All blots represent at least three independent in vitro experiments.
7
Histological Analysis of BP Scaffolds
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BP scaffolds with/without hMSC seeding were histologically processed using hematoxylin and eosin (H&E), immunohistochemistry, and immunofluorescence double staining. Briefly, the scaffolds were fixed in 10% buffered formalin, embedded in paraffin, and processed for staining. Laminin immunohistochemistry was performed using anti-laminin primary antibody (1:50, Abcam, Cambridge, MA) and horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (DAKO, Carpinteria, CA). Immunofluorescence double staining was performed using mouse anti-eGFP (1:200, Abcam, Cambridge, MA) and rabbit anti-laminin primary antibodies. Fluorescent anti-mouse secondary antibody and anti-rabbit secondary antibody tagged with Alexa Fluor 405 (402Ex/421Em) and Alexa Fluor 647 (652Ex/668Em) (1:500, Abcam), respectively, were used for visualization. For both stains, slides were imaged using a Nikon Eclipse E600 microscopy and digital images collected.
8
Protein Expression Analysis in Synovium
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The total synovial proteins were extracted using the protein extraction kit (Beibo Corp., Shanghai, China) and then resolved by electrophoresis. Next, the proteins were transferred from the gel onto the PVDF membrane (Millipore Corp., USA) using the semidry method (Tianneng Corp., China). The PVDF membranes were incubated with respective MAPK, PI3K, Akt1,PTEN,p-MAPK, p-PI3K, p-Akt1, and p-PTEN antibodies (dilution 1 : 1000; Abcam Biotechnology, Cambridge, UK) for 2 h at RT, followed by incubation with the following secondary antibodies for 1 h at RT: antimouse secondary antibody (dilution 1 : 5000. Zhongshan Goldenbridge Biotechnology Corp., Beijing, China) for MAPK, PI3K, Akt1, PTEN, p-MAPK, p-PI3K, p-Akt1, and p-PTEN. The enhanced chemiluminescence method (Thermo) was used to develop the bands, and the images were obtained.
9
Evaluating Axonal Damage and Astrocytic Response in EAE Models
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Spinal cord sections from EAE models (at 19 dpi, n = 6 in each group) were stained with neurofilament (NF) mouse mAb (Cell Signaling Technology, Beverly, MA, USA) followed by anti-mouse secondary antibody (Abcam (Hong Kong) Ltd, New Territories, HK) for evaluating axonal damage. Moreover, spinal cord sections and astrocytes cultured in vitro were dually stained with mouse anti-mouse GFAP (Millipore, Bedford, MA, USA) and rabbit anti-mouse CCL20 (Abcam (Hong Kong) Ltd, New Territories, HK), followed by incubation with anti-mouse and anti-rabbit secondary antibodies (Abcam (Hong Kong) Ltd, New Territories, HK). Stained sections were examined and photographed using a Leica DMI 4000B microscope (Leica Corp., Lasertechnik, Heidelberg, Germany) (for spinal cord sections) or Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany) (for astrocytic cultures). Mean fluorescence intensity was calculated using Image Pro Plus (Media Cybernetics, Silver Spring, MD, USA).
10
Western Blot Analysis of Cell Lysates
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Cells were lysed with RIPA buffer (CST, Danvers, MA, USA), and 45 ug proteins were run on an 8% SDS-polyacrylamide gel and then transferred onto the PVDF membrane. After 1 hour of blocking with 5% skim milk, the membranes were incubated with primary antibodies overnight at 4°C. The corresponding secondary antibodies were incubated at room temperature for 1 hour the next day. Primary antibodies against IGF-1 (DF6096, 1 : 500), vimentin (AF7013, 1 : 2000), E-cadherin (AF0131, 1 : 500), and β-actin (AF7018, 1 : 2000) were purchased from Affinity Biosciences (Jiangsu, China), with β-actin as an internal control. Subsequently, we washed the membranes (three times for 15 min, each wash) with TBST and incubated them with a goat antirabbit (1 : 3000, Abcam, Cambridge, MA, USA) or antimouse secondary antibody (1 : 3000, Abcam) for 2 h at 26 ± 2°C. An Odyssey infrared imaging system (Odyssey CLx, Biosciences, USA) was used to detect immunoreactivity.
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