Horseradish peroxidase hrp conjugated secondary antibody

Urine samples collected from the ureters of mice were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). Membranes were blocked, washed, and incubated overnight at 4°C with primary antibodies to mouse Kim-1 (Abcam, Cambridge, UK) and mouse Ngal (Santa Cruz Biotechnology, Avenue, CA, USA). The bound antibodies were detected on X-ray film by using enhanced chemiluminescence with horseradish peroxidase (HRP)-conjugated secondary antibodies and cyclic diacylhydrazides (Merck Millipore).

Total protein was extracted from tissues, resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and transferred to a polyvinylidene difluoride (PVDF) membrane (EMD Millipore, Danvers, MA, USA). After blocking, the membranes were incubated overnight with antibodies against α-tubulin (GeneTex, Irvine, CA, USA), type I collagen (COLA-1; ABclonal, Woburn, MA, USA), type III collagen (COLA-3; ABclonal), fibronectin (GeneTex), NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3; Proteintech, Rosemont, IL, USA), caspase-1 (Proteintech), interleukin-1β (IL-1β; Cell Signaling Technology, Danvers, MA, USA), interleukin-18 (IL-18; Proteintech), and gasdermin D (GSDMD; Santa Cruz Biotechnology, Dallas, TX, USA). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (EMD Millipore) for 1 h. Protein bands were visualized with enhanced chemiluminescence (ECL) reagents (EMD Millipore) and quantified using densitometry with Image J software (v1.46). The band intensities of the proteins of interest were normalized by that of α-tubulin or the uncleaved band.

Proteins were resolved in 12% polyacrilammide gels and blotted onto nitrocellulose filters (GE Heath Care, Little Chafont, Buckinghamshire, UK) for 2 hours at 250 mA on ice. Filters were blocked in phosphate buffered saline plus 0.1% Tween-20 (PBST, SIGMA) added with 10% non-fat dry milk, for 1 hour at room temperature (RT). Primary antibodies were incubated O/N at 4 °C, according to the concentration recommended by the manufacturer, in PBST plus 2.5% non-fat dry milk. After three 5 minutes washes, filters were incubated for 1 hour at RT with either goat-anti rabbit (1:5000) or goat-anti mouse (1:2000) horseradish peroxidase (HRP)-conjugated secondary antibodies (Merck Millipore, Darmstadt, Germany), or with ImmunoCruz F or C detection reagent, in the case of immunoprecipitated proteins, in PBST plus 2.5% non-fat dry milk. Proteins were revealed by SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). Images were captured with a Chemidoc XRS⁺ (Bio-Rad, Hercules, CA, USA).
Anti-H4R3me2s, anti-MEP50 and anti-PRMT5 antibodies were from Abcam (Abcam, Cambridge, UK); anti-Flag and anti-H3 antibodies were from SIGMA. Anti-H3K4me3, anti-H3K27me3 and anti-H3K9Ac antibodies were from Merck-Millipore. Anti-Myc and anti-TATA Binding Protein (TBP) were from Santa Cruz Biotechnologies.

Tissue or cell lysates were separated by SDS-PAGE gel and transferred to PVDF membrane (Millipore). After blocking with 5% milk, the same antibodies used for VEGF and osteocalcin IHC were used for WB detection. Signals were developed by film after incubating with appropriate horseradish peroxidase (HRP) - conjugated secondary antibodies (Millipore). Densitometry quantification was performed using software in BioSpectrum Imaging System (UVP, Upland, CA, USA). Intensity for all selected bands was all normalized to their actin (loading control) intensity then calculated as fold change to vector group. Data from three independent repeat experiments were used for statistical analysis.

Three days after MCAO in rats, tissues from the SVZ and ischemic penumbra were harvested. The tissues were lysed in lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor followed by centrifugation for 15 min at 14,000 rpm. The total protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked and incubated with an appropriate primary antibody and secondary antibody. The following antibodies were used: mouse anti-GAPDH (1:5,000, CST, Danvers, MA, USA), rabbit anti-PKC (1:500, Wanleibio, Shen Yang, China), rabbit anti-JNK (1:500, Wanleibio, Shen Yang, China), rabbit anti-β-catenin (1:10,000, Proteintech Group, Chicago, IL, USA), rabbit anti-p-PKC (1:10,00, CST, Danvers, MA, USA), rabbit anti-p-JNK (1:500, Wanleibio, Shen Yang, China), and horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; Millipore, Billerica, MA, USA).