Immunohistochemistry was carried out using a two-step detection kit (Zhongshan Golden Bridge Biotechnology, China). Briefly, specimens were immersed in antigen retrieval solution for 20 min, blocked for 30 min with 5% bovine serum albumin (BSA), and subsequently incubated with primary antibodies against mouse TNF-α (Abcam, ab1793) and IL-6 (Abcam, ab290735) for inflammatory cytokines markers, CD31 (Abcam, ab182981) and TGF-β (Abcam, ab215715) for promoting repair markers at 1:100 dilution overnight at 4 °C. After rinsing thoroughly in PBS, the horseradish peroxidase-conjugated secondary antibodies (Zhongshan Golden Bridge Biotechnology) were dropped onto slides. Each group is composed of more than three slides, and each slide was observed using a Zeiss light microscopy at the defect area including the scaffolds (N > 3).
Horseradish peroxidase conjugated secondary antibody
Manufactured by Zhongshan Golden Bridge Biotechnology
Sourced in China
Horseradish peroxidase-conjugated secondary antibody is a laboratory reagent used in various immunological techniques. It consists of a secondary antibody that is chemically conjugated to the enzyme horseradish peroxidase. This enzyme-linked antibody can be used to detect and visualize target proteins or antigens in samples.
Automatically generated - may contain errors
Lab products found in correlation
Sox9 antibody, by Merck Group (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
β actin antibody, by Santa Cruz Biotechnology (2 mentions)
γh2ax, by Cell Signaling Technology (2 mentions)
Alexa fluor 488 conjugated antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 conjugated antibody, by Thermo Fisher Scientific (2 mentions)
Sycp3, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 488 conjugate of lectin pna, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Zhongshan Golden Bridge Biotechnology (2 mentions)
Anti β actin, by Zhongshan Golden Bridge Biotechnology (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab137494, by Abcam (2 mentions)
Ab215715, by Abcam (1 mentions)
32 protocols using horseradish peroxidase conjugated secondary antibody
1
Immunohistochemical Analysis of Inflammatory and Repair Markers
2
Investigating Matrix Metalloproteinase Regulation
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Bleomycin was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. (Taizhou, China). Artesunate was purchased from Guilin Pharmaceutical Co., Ltd. (Guilin, China). Primers were synthesized by Invitrogen Life Technologies (Shanghai, China). The cDNA synthesis kit was purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The total RNA extraction kit and 2x Taq PCR Master mix were obtained from Tiangen Biotech Co., Ltd. (Beijing, China). The TIMP-1 and TIMP-2 antibodies and horseradish peroxidase-conjugated secondary antibodies were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). Collagen-IV antibody was purchased from Abcam Ltd. (Hong Kong, China). MMP-2 antibody and MMP-9 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Hong Kong, China).
3
Immunofluorescence Staining of Testicular Germ Cells
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β-actin antibody (mouse, sc-47778; Santa Cruz); SYCP3 (mouse, sc-74569; Santa Cruz); γH2AX (rabbit, 9718; Cell Signaling Technology, Inc.); MVH (mouse, ab27591; Abcam); SOX9 antibody (rabbit, AB5535, Sigma-Aldrich); PLZF antibody (goat, AF2944, R&D Systems); SFRS1 polyclonal antibody (rabbit, 12929-2-AP, Proteintech); c-Kit antibody (goat, AF1356, R&D Systems). Horseradish peroxidase-conjugated secondary antibodies were purchased from Zhongshan Golden Bridge Biotechnology Co, LTD (Beijing). Alexa Fluor 488-conjugated antibody, 594-conjugated antibody and Alexa Fluor 647-conjugated antibody were purchased from Life Technologies.
4
Immunostaining Assay for Germ Cell Markers
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Β-actin antibody (mouse, sc-47778; Santa Cruz); SYCP3 (mouse, sc-74569; Santa Cruz); γH2AX (rabbit, 9718; Cell Signaling Technology, Inc.); MVH (mouse, ab27591; Abcam); SOX9 antibody (rabbit, AB5535, Sigma-Aldrich); PLZF antibody (goat, AF2944, R&D Systems); SFRS2 polyclonal antibody (rabbit, 20,371–1-AP, Proteintech); SC35 antibody (mouse, S4045, Sigma-Aldrich); green-fluorescent Alexa Fluor® 488 conjugate of lectin PNA (L21409, Thermo). Horseradish peroxidase–conjugated secondary antibodies were purchased from Zhongshan Golden Bridge Biotechnology Co, LTD (Beijing). Alexa Fluor 488–conjugated antibody, 594–conjugated antibody and Alexa Fluor 647–conjugated antibody were purchased from Life Technologies.
5
Western Blotting of Cell Signaling Proteins
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Western blotting was performed as described by Yang et al. (Yang et al., 2012 (link)). Briefly, cells were washed with cold PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer (high) (R0010, Solarbio) containing a protease inhibitor cocktail (P8340, Merck Millipore) and phosphatase inhibitor cocktails II and III (HY-K0022, HY-K0023, MedChem Express). Thirty micrograms of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) followed by electrotransfer onto nitrocellulose membranes. After blocking with 5% skim milk for 1 h, membranes were incubated with primary antibodies against proliferating cell nuclear antigen G-CSF (1:200; #bs-1023R; Bioss), G-CSFR (1:300; #bs-2574R; Bioss), p-PI3K (1:300; #4228; Cell Signaling Technology), PI3K (1:300; #4249; Cell Signaling Technology), p-Akt (1:300; #4060; Cell Signaling Technology), Akt (1:500; #4691; Cell Signaling Technology), or GAPDH (1:2000; #ab22556; Abcam) overnight at 4°C. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Zhongshan Golden Bridge Biotechnology Co., Ltd) for 1 h at room temperature, and signals were developed using an enhanced chemiluminescence system (Piece). Densitometric quantification was performed using Scion Image software (Scion Corp.).
6
Immunostaining Antibody Inventory for Germ Cell Research
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PPP6C antibody (rabbit, A300-844A; Bethyl Laboratories, Inc.); SYCP3 antibody (rabbit, NB300-231; Novus Biologicals); α-tubulin antibody (rabbit, 2144; Cell Signaling Technology, Inc.); β-actin antibody (mouse, 3700; Cell Signaling Technology, Inc.); Phospho-β-Catenin (Ser552) (D8E11) antibody (rabbit, 5651; Cell Signaling Technology, Inc.); SYCP3 antibody (mouse, sc-74569; Santa Cruz); γH2AX antibody (rabbit, 9718; Cell Signaling Technology, Inc.); MVH antibody (mouse, ab27591; abcam); SYCP1 antibody (rabbit, ab15090; abcam); SOX9 antibody (rabbit, AB5535, Sigma-Aldrich); PLZF antibody (goat, AF2944, R&D Systems); β-catenin antibody (rabbit, 51067-1-AP, Proteintech); ZO2 antibody (rabbit, 18900-1-AP, Proteintech); TEX14 antibody (rabbit, 18351-1-AP, Proteintech); Vimentin antibody (rabbit, 10366-1-AP, Proteintech); HA-Tag mab (mouse, AE008; ABclonal); c-Myc antibody (mouse, m4439; sigma); green-fluorescent Alexa Fluor® 488 conjugate of lectin PNA (L21409, Thermo). Horseradish peroxidase–conjugated secondary antibodies were purchased from Zhongshan Golden Bridge Biotechnology Co, LTD (Beijing). Alexa Fluor 488–conjugated antibody and Alexa Fluor 594–conjugated antibody were purchased from Life Technologies.
7
Protein Expression Profiling via Western Blot
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Protein extracts were prepared and resolved on 12% SDS-PAGE gels. After transferring the proteins to nitrocellulose membranes, the membranes were blocked with 5% non-fat milk in PBS for 2 h and then incubated separately with primary antibodies against GAPDH, P53, AMPK, P-AMPK, mTOR, c-Myc, Sox-2, P-P65, P65, IKBα, P-IKBα (Cell Signaling, City, MA, USA), Bax or Bcl-2 (Boster Corporation, Wuhan, China) followed by incubation with horseradish peroxidase-conjugated secondary antibodies (ZhongShan Golden Bridge Biotechnology, Beijing, China). Antibodies were detected using enhanced chemiluminescence reagent (Pierce Biotechnology, City, IL, USA).
8
TAZ Expression in OSCC Cell Lines
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TAZ expression in OSCC cell lines (SCC9, HSC3, SCC15) was detected by protein immunoblot. Cells were lysed in RIPA buffer (Beyotime, China) in the presence of protease inhibitor and phosphatase inhibitor. Equivalent amounts of cell extracts were separated using SDS-PAGE, and transferred to polyvinylidene fluoride membrane, followed by immunoblotting hybridization using TAZ antibody (Abcam, Cambridge, MA, USA). Bands were visualized using the enhanced chemiluminescene system (Millipore, USA). For the SCC9 western blot assay, cells were treated with AR-42 for 24 h, and then lysed with RIPA buffer. The proteins of interest were detected on whole-cell extracts as described above. The primary antibodies were all purchased from Abcam and Cell Signaling Technology, and used at a 1:1,000 dilution. The horseradish peroxidase-conjugated secondary antibodies (Zhong Shan Golden Bridge Bio-technology, China) were used at 1:5,000.
9
Western Blot Analysis of NF-κB Pathway
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After treatment as indicated in the legends, total cell protein was extracted using RIPA buffer (ShangHai Biocolor BioScience Technology Company, Shanghai, China) containing 1/10 volumn of PhosSTOP Phosphatase inhibitor Cocktail (Roche), and Western blot analysis was performed as previously described [37 (link)]. The following primary antibodies were used with 1:2000 dilution: phosphorylated-NF-κB p65 (p-p65; Ser536; 93H1), NF-κB p65 (D14E12) XP® (Cell Signaling Technology, Inc., Beverly, MA), and GAPDH (Zhongshan Goldenbridge Biotechnology Co., Ltd. Beijing, China). Immunocomplexes on PVDF membrane were detected with appropriate horseradish peroxidase–conjugated secondary antibodies (1:2000; Zhongshan Goldenbridge). The membranes were developed with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) according to manufacturer’s instructions and the pictures were collected using GeneGnome5 (Gene Company, Ltd. Hong Kong, China).
10
Protein Extraction and Western Blot Analysis
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Total protein was extracted from human tissues by using RIPA Lysis Buffer (Beyotime). The protein concentration was determined by a BCA Protein Assay Kit (Bio‐Rad). Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was used, and equal amounts of protein (20 µg) were added to each electrophoresis well on the gel plate. Next, a semidry electroblotting system (Bio‐Rad) was used to transfer the electrophoretic proteins onto a polyvinylidene fluoride membrane (Millipore). The blots were incubated in Tris‐buffered saline with Tween (TBST, 20 mmol/L Tris‐HCl, pH 8.0, 150 mmol/L NaCl, 0.5% Tween‐20) and 5% skim milk for 2 h at 37°C. Then, the blots were incubated for 12 h at 4°C in TBST containing primary antibodies against FPR2 (1:800; Abcam), NF‐κB (1:500; Cell Signaling Technology), and GAPDH (1:1000; Cell Signaling Technology). After the blots were washed in TBST, they were incubated with horseradish peroxidase‐conjugated secondary antibodies (1:1000; Zhongshan Goldenbridge) for 2 h at 37°C. The protein bands on the membranes were observed with enhanced chemiluminescence, and densitometry was calculated using ImageJ software (National Institutes of Health). GAPDH levels were evaluated as a loading control.
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