The hippocampus was dissected out from APP/PS1 and APP/PS1 TBI mice. Western blotting was performed as described previously (30 (link)). Target protein expression was measured using the following primary antibodies: rabbit anti-arginase-1 (Arg1) (catalog number: #93668; Cell Signaling Technology), rabbit anti-inducible nitric oxide synthase (iNOS) (catalog number: #13120; Cell Signaling Technology), and rabbit anti-β-actin (catalog number: #4790; Cell Signaling Technology) (IgGs; 1:2000). β-Actin was utilized as an internal reference. The protein blots were visualized using an HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:5000, Invitrogen; Thermo Fisher Scientific) and a Chemi-Doc™ Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and analyzed using Image-Pro Plus 6.0.
Hrp conjugated goat anti rabbit igg h l secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody is a laboratory reagent used to detect and quantify rabbit primary antibodies in various immunoassays. It is a polyclonal antibody produced in goats and conjugated with horseradish peroxidase (HRP) enzyme, which can be used for signal amplification and visualization.
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Lab products found in correlation
Image lab software, by Bio-Rad (3 mentions)
Beyoecl plus, by Beyotime (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Hrp conjugated goat anti mouse igg h l secondary antibody, by Thermo Fisher Scientific (2 mentions)
Non fat milk powder, by Beyotime (2 mentions)
Rabbit anti inducible nitric oxide synthase, by Cell Signaling Technology (1 mentions)
Image pro plus 6, by Bio-Rad (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Beyoecl star, by Beyotime (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Alexa fluor 647 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Anti pan adp ribose binding reagent, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Tetro cdna synthesis kit, by Meridian Bioscience (1 mentions)
11 protocols using hrp conjugated goat anti rabbit igg h l secondary antibody
1
Quantifying Hippocampal Inflammation in AD/TBI Mice
2
Quantitative Protein Analysis Pipeline
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To isolate and quantify the total protein, the RIPA Lysis Buffer (Beyotime, China) and Enhanced BCA Protein Assay Kit (Beyotime, China) were adopted, respectively. Then, the sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out with 4%–20% precast gel (Willget, China). Subsequently, the protein was transferred to polyvinylidene fluoride membrane (PALL, Germany), followed by incubating with JKAP polyclonal antibody (1: 1500) (Invitrogen, USA) and HRP‐conjugated goat anti‐rabbit IgG (H + L) secondary antibody (1:50,000), sequentially. At last, BeyoECL Star (Beyotime, China) was used to visualize the protein bands.
3
Immunoblotting and Immunofluorescence Techniques
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Anti-Myc (2276S), anti-β-actin (8457S), anti-GluR1 (13185S), anti-NR1 (5704S), anti-PSD-95 (3450S), and anti-Map2 (4542S) antibodies are from Cell Signaling Technology. Anti-GFAP (16825-1-AP), anti-GluR2 (11994-1-AP), anti-NR2A (19953-1-AP), and anti-NR2B (19954-1-AP) antibodies are from Proteintech. Anti-Iba1 (019-19741), anti-GFP (M20004L), anti-NeuN (ab177487), anti-GluR3 (MAB5416), and anti-synaptophysin (S5768) antibodies are from Wako, Abmart, Abcam, Millipore, and Sigma, respectively. HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (31430), HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (31460), Alexa fluor 488-conjugated donkey anti-mouse IgG (H + L) secondary antibody (A-21202), Alexa fluor 488-conjugated goat anti-rabbit IgG (H + L) secondary antibody (A-11008), and Alexa fluor 594-conjugated goat anti-rabbit IgG (H + L) secondary antibody (A-11012) are from Thermo Fisher Scientific.
4
Immunofluorescence Staining Reagents
5
Quantifying Target Gene Expression
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Total RNA was isolated from cultured cells using the High Pure RNA Isolation Kit (Roche, Basel, Switzerland). RNA yield and purity were measured on a NanoDrop spectrophotometer (NanoDrop Technologies, Thermo Fisher).
1 μg of RNA was subsequently reverse transcribed with the Tetro cDNA Synthesis Kit (Bioline, Meridian Bioscience, Cincinnati, OH). Real-Time PCR was performed in a thermal cycler (StepOnePlus, Applied Biosystems, Thermo Fisher) with SensiFast SYBR Hi-ROX reagent (Bioline) capturing target genes FARSA, FARSB, and the ribosomal internal control gene RPS29 (primers, see supplementary information S1). Fold change of target gene expression was calculated based on the comparative C T method. 24 (link) NuPage Mini 3%-8% Tris-Acetate gel (Thermo Fisher) and blotted to a polyvinylidene fluoride membrane (Merck). Antigen targets were detected using the rabbit polyclonal primary antibodies anti-FARSA 18121-1-AP (Proteintech, St. Leon-Rot, Germany), and anti-FARSB HPA036677 (Sigma-Aldrich), followed by incubation with HRPconjugated goat anti-rabbit IgG (H + L) secondary antibody (Thermo Fisher). Β-actin quantification (sc-47 778 HRP, Santa Cruz, Dallas, TX) served as a loading control. Proteins were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher). Relative band intensities were analyzed using the Image J software. 25 (link)
1 μg of RNA was subsequently reverse transcribed with the Tetro cDNA Synthesis Kit (Bioline, Meridian Bioscience, Cincinnati, OH). Real-Time PCR was performed in a thermal cycler (StepOnePlus, Applied Biosystems, Thermo Fisher) with SensiFast SYBR Hi-ROX reagent (Bioline) capturing target genes FARSA, FARSB, and the ribosomal internal control gene RPS29 (primers, see supplementary information S1). Fold change of target gene expression was calculated based on the comparative C T method. 24 (link) NuPage Mini 3%-8% Tris-Acetate gel (Thermo Fisher) and blotted to a polyvinylidene fluoride membrane (Merck). Antigen targets were detected using the rabbit polyclonal primary antibodies anti-FARSA 18121-1-AP (Proteintech, St. Leon-Rot, Germany), and anti-FARSB HPA036677 (Sigma-Aldrich), followed by incubation with HRPconjugated goat anti-rabbit IgG (H + L) secondary antibody (Thermo Fisher). Β-actin quantification (sc-47 778 HRP, Santa Cruz, Dallas, TX) served as a loading control. Proteins were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher). Relative band intensities were analyzed using the Image J software. 25 (link)
6
SARS-CoV-2 Spike Protein ELISA
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Serum from the rabbit of different groups were analyzed by enzyme‐linked immunosorbent assay (ELISA) to determine sera antibody titers. ELISA plate (Corning, USA) was coated with 1 µg/ml SARS-CoV-2 Spike S1 + S2 ECD-His recombinant protein (Sino Biological, China) in Dulbecco’s phosphate-buffered saline (DPBS) (ThermoFisher, USA) for 2 h at room temperature. Plate was washed for 3 times with DPBS + 0.05% Tween 20 (Scharlau, Spain) and then blocked with PBS + 1% BSA (ThermoFisher, USA) + 0.050% Tween-20 for 2 h at 37 °C. The plate was washed for 3 times then incubated with rabbit sera and SARS-CoV-2 Spike antibody (Sino Biological, China) for 2 h at 37 °C. After washing for 3 times, the plate was incubated with HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (ThermoFisher, USA) for 50 min at room temperature. Final washing was done for 3 times and then developed for colorimetric reaction with Pierce TMB substrate (ThermoFisher, USA) for 10 min. The reaction was stopped with 1 N HCl and the plate was read at 450 nm wavelength within 30 min.
7
Cardiac Protein Quantification and Analysis
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Heart tissue and primary rat cardiomyocytes were lysed in RIPA lysis buffer with 1% protease inhibitor (Beyotime Institute of Biotechnology). A BCA kit was used to determine the protein concentration in the supernatant (Beyotime Institute of Biotechnology). In total, ~50 µg heart tissue lysate or 20 µg cell lysate was used for 12% SDS-PAGE, and proteins were then transferred to an FL membrane (MilliporeSigma) at 4˚C for 1.5 h. After blocking with 5% non-fat milk powder (Beyotime Institute of Biotechnology) at room temperature for 1.5 h, the following primary antibodies were added and incubated overnight at 4˚C: SOD2 (1:1,000), pro/cleaved caspase-3 (1:1,000), Bax (1:1,000), NOX2 (1:2,000) and Bcl-2 (1:1,000) were. Subsequently, HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibodies (1:10,000; Thermo Fisher Scientific, Inc.; cat. no. 31460) were added and incubated at room temperature for 1.5 h. The western blotting results were analyzed using BeyoECL Plus (Beyotime Institute of Biotechnology) and Image Lab software (version 5.2.1; Bio-Rad Laboratories, Inc.). The specific protein expression levels were normalized to that of GAPDH.
8
Cardiac mitochondrial protein analysis
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The mitochondria and cytoplasm of rat ventricular tissue and primary rat cardiomyocytes were separated according to the instructions of tissue or cell mitochondrial extraction kit (Beyotime Institute of Biotechnology). A BCA kit was used to determine the protein concentration thereafter. Then, ~30 µg heart tissue or cell lysate was loaded per lane onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, after which proteins were transferred to an FL membrane (MilliporeSigma) at 4˚C for 2 h. After blocking with 5% blocking protein powder (non-fat milk powder; Beyotime Institute of Biotechnology) at room temperature for 2 h, the following primary antibodies were added and incubated overnight at 4˚C: Cyt C (1:1,000), pro/cleaved caspase-3 (cat. no. 14220S/cat. no. 9664S; 1:1,000), cleaved caspase-9 (cat. no. 9507; 1:1,000), COX IV (cat. no. 4850; 1:1,000) and GAPDH (cat. no. 10494-1-AP; 1:5,000). Subsequently, HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibodies (1:10,000; Thermo Fisher Scientific, Inc.; cat. no. #31460) were added and incubated at room temperature for 1.5 h. The results of western blotting were analyzed using BeyoECL Plus (Beyotime Institute of Biotechnology) and Image Lab software (version 5.2.1; Bio-Rad Laboratories, Inc.). The specific protein expression levels were normalized to GAPDH or COX IV.
9
Western Blot Analysis of Cardiac Proteins
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Cardiac tissue and H9C2 cardiomyocytes were lysed in radioimmunoprecipitation lysis buffer containing 1% protease inhibitor to obtain a pure protein solution. Then, a BCA kit was used to determine the protein concentration. In total, approximately 40 μg of heart tissue lysate or 20 μg of cell lysate was used for 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and then, the proteins were transferred to PVDF membranes. After blocking with rapid protein-blocking solution, the following primary antibodies were added and incubated: RNF10 (1 : 1000), AP-1 (1 : 1000), Mexo2 (1 : 1000), cleaved caspase-3 (1 : 1000), Bax (1 : 1000), Bcl-2 (1 : 1000), and GAPDH (1 : 5000). Subsequently, membranes were added and incubated in HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibodies (1 : 10,000; Thermo Fisher Scientific, Inc.; 31460). BeyoECL Plus (Beyotime Institute of Biotechnology) and Image Lab software (version 5.2.1; Bio-Rad Laboratories, Inc.) were used to analyze protein expression. The specific protein expression levels were normalized to GAPDH.
10
Quantifying Exosomal Protein Expression
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EV protein extracts (300 μg per medium type) were separated by Mini-PROTEAN TGXPrecast Gels (BioRad) and transferred to PVDF membranes by using Trans-Blot Turbo RTA Mini PVDF Transfer Kit (BioRad). The expression level of Syntenin and CD63 was evaluated by using goat polyclonal IgG Syntenin/SDCBP antibody (PA5-18595, Invitrogen/Thermo Fisher Scientific) and mouse monoclonal IgG exosome—anti-CD63 antibody (10628D, Invitrogen), respectively. An equal loading in the lanes was evaluated by mouse monoclonal IgG β-actin antibody (sc-81178, Santa Cruz Biotechnology). The level of analyzed proteins was subsequently detected with horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG (H+L) secondary antibody (R21459, Invitrogen) or goat anti-mouse IgG, IgM (H+L) secondary antibody (31,444, Invitrogen). All antibodies were used according to manufacturer’s protocols. The membranes were developed with Luminata Crescendo Western HRP Substrate (Merck/Millipore, Darmstadt, Germany) and imaged by Gel Doc XR+ Gel Documentation System (Bio-Rad).
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