NSCLC cancerous and matched adjacent non-cancerous tissues were prepared for TMAs. The manual Tissue Microarray System Quick-Ray (UT06, UNITMA, Korea) was used to generate TMAs. Core tissue samples (diameter of 2 mm) were obtained from paraffin embedded tissue sections and deposited in paraffin-recipient blocks. Before immunohistochemical processing, the tissue microarray specimens were cut into 4-μm-thick sections and placed on the super frost charged glass microscope slides. IHC was carried out as described before [50 (link)]. Briefly, the slides were incubated with the primary antibodies for FABP3 (1:100; Abcam, Cambridge, MA, USA) and FABP4 (1:100; Abcam, Cambridge, MA, USA) overnight at 4°C. Subsequently, the slides were incubated with anti-mouse IgG secondary antibody (Abcam) for FABP3 and horseradish-peroxidase-conjugated rabbit IgG (Abcam) for FABP4. Diaminobenzidine solution was used for the color development.
Secondary anti mouse igg antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Secondary anti-mouse IgG antibody is a laboratory reagent used to detect the presence of mouse immunoglobulin G (IgG) in samples. It binds to the Fc region of mouse IgG, allowing for visualization or quantification of target proteins in various immunoassays and techniques.
Automatically generated - may contain errors
Lab products found in correlation
Tecnai g2 f20 s twin transmission electron microscope, by Thermo Fisher Scientific (3 mentions)
H7000 tem, by Hitachi (3 mentions)
Paraformaldehyde solution, by Santa Cruz Biotechnology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (2 mentions)
Fluoview fv10i confocal microscope, by Olympus (2 mentions)
Alkaline phosphatase, by Bio-Rad (2 mentions)
Fabp4, by Abcam (1 mentions)
Horseradish peroxidase conjugated rabbit igg, by Abcam (1 mentions)
Anti rat nlrp3 antibody, by Abcam (1 mentions)
Triton, by Merck Group (1 mentions)
P jak2, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Nf κb, by Abcam (1 mentions)
Bicinchoninic acid kit, by Merck Group (1 mentions)
P akt, by Abcam (1 mentions)
P pi3k p85, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
13 protocols using secondary anti mouse igg antibody
1
NSCLC Tissue Microarray Immunohistochemistry
2
Immunofluorescence Analysis of NLRP3 in Chondrocytes
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The chondrocytes, cultured on cover slips for 48 h, were fixed with 4 % paraformaldehyde solution (Santa Cruz Biotechnology, USA), followed by treatment with blocking solution containing 5 % donkey serum and 0.1 % Triton (Sigma Aldrich, Germany) in PBS. Subsequently, the samples were incubated with the polyclonal anti-Rat NLRP3 antibody (1:60, abcam, USA) overnight at 4 °C followed by washing three times with PBS and incubating with the anti-mouse IgG secondary antibody (1:200, abcam, USA) and DAPI (Roche Applied Science, Germany) for 1 h at room temperature. The images were collected by an Olympus FluoView FV10i confocal microscope.
3
Immunofluorescence Staining of Chondrocytes
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After a 48-h incubation on coverslips, chondrocytes were fixed using a 4% paraformaldehyde solution provided by Santa Cruz Biotechnology, USA. They were then incubated in a blocking solution containing 0.1% Triton X-100 (sourced from Sigma Aldrich, Germany) in PBS solution Overnight incubation at 4 °C followed, during which the samples were treated with polyclonal antibodies anti-Rat NLRP3, NF-κB, p-JAK2, or N-GDSMD at a dilution of 1:60 (Abcam, USA). After thrice washing with PBS, the specimens were incubated at 37 °C for 1 h with an anti-mouse IgG secondary antibody at a dilution of 1:200 (Abcam, USA) and DAPI (acquired from Roche Applied Science, Germany) for nuclear staining. The resulting images were captured using an Olympus FluoView FV10i confocal microscope.
4
Western Blot Analysis of Apoptosis Markers
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Total protein was extracted from PC12 cells using pre-cold RIPA buffer (Beyotime Institute of Biotechnology) including protease inhibitor. Total protein was quantified using a bicinchoninic acid kit (Sigma-Aldrich; Merck KGaA). Proteins (40 µg) were then separated via 10% SDS-PAGE, proteins were transferred onto PVDF membranes. The membranes were then blocked with 5% skimmed milk at room temperature for 1 h. The membranes were incubated overnight at 4˚C with anti-PDCD4 (1:1,000; cat. no. ab80590; Abcam), PI3K (1:1,000; cat. no. ab32089; Abcam), p-PI3K p85 (1:1,000; cat. no. ab278545; Abcam), AKT (1:1,000; cat. no. ab8805; Abcam), p-AKT (1:1,000; cat. no. ab81283; Abcam), Bax (1:1,000; cat. no. ab32503; Abcam), caspase-3 (1:1,000; cat. no. ab13847; Abcam) and GAPDH (1:1,000; cat. no. ab9485; Abcam) primary antibodies in the absence of light. Then cells were incubated with HRP-labeled anti-rabbit IgG secondary antibody (1:4,000; cat. no. ab6721; Abcam) or anti-mouse IgG secondary antibody (1:5,000, cat. no. ab6789, Abcam) for 2 h at room temperature. The bands were imaged with an Pierce™ ECL Western Blotting Substrate kit (cat. no. 32109; Thermo Fisher Scientific, Inc.) and band density was quantified using Quantity One v.6.2 software (Bio-Rad Laboratories, Inc.).
5
Colorectal Cancer Tissue Microarray Evaluation
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Tissue microarray slides were obtained from Outdo Biotech (Shanghai, China), which contained 90 pairs of adjacent paracancer tissues and colorectal cancer tissues. The slides were stained with primary antibodies against β3GnT8, CD147 (Santa Cruz Biotechnology, Dallas, TX, United States), galectin3 (Abcam, Cambridge, MA, United States), MMP-2 (Abcam), β3GnT2 (Santa Cruz Biotechnology), and HRP-conjugated anti-rabbit IgG, or anti-mouse IgG secondary antibody (Abcam). The protein expression was detected by DAB horseradish peroxidase color development kit (Beyotime, Haimen, China). The slides were evaluated by the staining intensity and positive cells percentage as follows: staining intensity, 0(no), 1(weak), 2(moderate), 3(strong), and positive cells percentage, 0(<1%), 1(1–33%), 2(34–66%), 3(67–100%). The final grade of target protein expression was calculated by plus the score of staining intensity and the score of positive cells percentage: 0(0), 1+(1–2), 2+(3–4), and 3+(5–6). The expression scores of β3GnT8, CD147, galectin3 and MMP-2 were provided in Supplementary Data Sheet 1 (Supplementary Tables 1–4 ).
6
Quantifying Phosphorylated PMK-1 in C. elegans
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C. elegans were prepared as described above to ensure that stage-matched, hermaphrodite animals at the young L4 larval stage were studied in each condition. Protein lysates were prepared as previously described [17 (link)] and probed with a 1:1000 dilution of an antibody that recognizes the doubly-phosphorylated TGY motif of PMK-1 (Promega Corporation). Monoclonal anti-α-tubulin antibody was used at a dilution of 1:1,000 (Sigma-Aldrich). A polyclonal antibody against the total PMK-1 protein was raised using the peptide DFQKNVAFADEEEDEEKMES (PMK-1 amino acids 358 to 377) in a rabbit (Thermo Scientific Pierce Custom Antibody Services) and used at a dilution of 1:1000. We confirmed that the total PMK-1 antibody detects total, but not active (phosphorylated) PMK-1 (Fig 5C ). Horseradish peroxidase (HRP)-conjugated anti-rabbit (Cell Signaling Technology) and anti-mouse IgG secondary antibodies (Abcam) were diluted 1:10,000 and used to detect the primary antibodies following the addition of ECL reagents (Thermo Fisher Scientific, Inc.), which were visualized using a BioRad ChemiDoc MP Imaging System. The band intensities were quantified using BioRad Image Lab software version 5.2.1, and the ratio of active phosphorylated PMK-1 to total PMK-1 was calculated with all samples normalized to the ratio of wild-type control animals.
7
Western Blot Analysis of Protein Markers
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Total protein samples were extracted from Hep3B and Bel7402 cells, separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were then blocked with non-fat milk (5% in TBST) at 4°C overnight, and the primary antibody was incubated for 2 h at room temperature. After washing three times with TBST (10 min for each time), the membranes were incubated with horseradish peroxidase (HRP) conjugated secondary antibodies for 2 h at room temperature. Blots were visualized with enhanced chemiluminescence reagents and analyzed using ImageJ.
The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): E-cadherin, PTEN, p-AKT (Ser473), AKT, p-mTOR, mTOR, p-p70S6K1 (Thr389), p70S6K1, p-ERK1/2, ERK1/2, Bcl-2, and Bax. The antibody against GAPDH was purchased from Proteintech (Rosemont, IL, USA). Anti-rabbit IgG and anti-mouse IgG secondary antibodies conjugated with horseradish peroxidase (HRP) were purchased from Abcam (Cambridge, UK).
The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): E-cadherin, PTEN, p-AKT (Ser473), AKT, p-mTOR, mTOR, p-p70S6K1 (Thr389), p70S6K1, p-ERK1/2, ERK1/2, Bcl-2, and Bax. The antibody against GAPDH was purchased from Proteintech (Rosemont, IL, USA). Anti-rabbit IgG and anti-mouse IgG secondary antibodies conjugated with horseradish peroxidase (HRP) were purchased from Abcam (Cambridge, UK).
8
Transmission Electron Microscopy of Amyloid and Polyglutamine Aggregates
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The end-point products of aggregated samples were placed on glow-discharged, 400-mesh Formvar carbon-coated copper grids (EMS Inc., Hatfield, PA, USA) for 3 min (Aβ or α-synuclein) or for 1 min (TrxHttex1-polyQ), rinsed, and negatively stained with 2% uranyl acetate (UA). Samples were examined in Hitachi H-7000 TEM (Hitachi Inc., Tokyo, Japan) at an accelerating voltage of 75 kV. For immunogold staining, the incubated TrxHttex1-39Q (50 μM) with and without equimolar SERF1a samples were dropped on the grids for 10 min and rinsed. After air-drying, the grids were then probed with primary SERF1a antibody (1:100; monoclonal antibody) and the 10 nm gold-conjugated secondary anti-mouse IgG antibody (1:500; abcam). The grids were finally stained with 1% UA. The samples were observed with a FEI Tecnai G2 F20 S-TWIN transmission electron microscope at 120 kV with TEM user interface and DigitalMicrograph software.
9
Transmission Electron Microscopy of Amyloid and Polyglutamine Aggregates
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The end-point products of aggregated samples were placed on glow-discharged, 400-mesh Formvar carbon-coated copper grids (EMS Inc., Hatfield, PA, USA) for 3 min (Aβ or α-synuclein) or for 1 min (TrxHttex1-polyQ), rinsed, and negatively stained with 2% uranyl acetate (UA). Samples were examined in Hitachi H-7000 TEM (Hitachi Inc., Tokyo, Japan) at an accelerating voltage of 75 kV. For immunogold staining, the incubated TrxHttex1-39Q (50 μM) with and without equimolar SERF1a samples were dropped on the grids for 10 min and rinsed. After air-drying, the grids were then probed with primary SERF1a antibody (1:100; monoclonal antibody) and the 10 nm gold-conjugated secondary anti-mouse IgG antibody (1:500; abcam). The grids were finally stained with 1% UA. The samples were observed with a FEI Tecnai G2 F20 S-TWIN transmission electron microscope at 120 kV with TEM user interface and DigitalMicrograph software.
10
Quantitative Sandwich ELISA for IL-5 and Antimyosin IgG
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Sera were stored at −80°C before analysis. IL-5 was determined by quantitative sandwich ELISA according to the manufacturer’s recommended protocols (R&D Systems). For antimyosin IgG ELISA, plates were coated with 0.5 µg/ml MyHC614629 peptide (AcSLKLMATLFSTYASAD; Genscript) overnight at 4°C, washed with 1× PBS and 2% FBS, and incubated with sera for 2 h at room temperature. Plates were washed and incubated with secondary anti–mouse IgG antibody (Abcam) diluted 1:1,000 in 1× PBS for 2 h at room temperature. Plates were washed and developed with alkaline phosphatase (Bio-Rad Laboratories), and OD was read at 405 nm.
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