The study was approved by the UCLA Institutional IRB Committee. Archived FFPE tissues of OLPs and OLP-OSCCs were used for liquid chromatography with tandem mass spectrometry (LC-MS/MS) and IHC. To perform IHC analysis, FFPE tissue sections were deparaffinized by sequential washing with xylene, 100% ethanol, 95% ethanol, 80% ethanol and PBS. The sections were incubated with 0.3% H2O2 in methanol for 5 min to quench the endogenous peroxidase activity. The slides were blocked in PBS with 5% BSA for 30 min and then incubated overnight with a 1:100 dilution of anti-SOX4 primary antibody (Abcam, Cambridge, MA, USA) at 4 °C. After sections were rinsed with PBS, they were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:2000; GE Healthcare, Piscataway, NJ, USA) for 2 h at room temperature.
Horseradish peroxidase hrp conjugated anti rabbit igg
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG is a laboratory reagent used in various immunoassay techniques. It consists of antibodies that bind specifically to rabbit immunoglobulin G (IgG) and are conjugated with the enzyme horseradish peroxidase. This conjugate can be used to detect and quantify the presence of rabbit IgG in biological samples.
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Lab products found in correlation
Rabbit anti gapdh, by Merck Group (2 mentions)
Imagequant las 4000 mini system, by GE Healthcare (2 mentions)
Anti actin, by Merck Group (2 mentions)
Anti mouse igg hrp, by GE Healthcare (2 mentions)
Mitochondria isolation kit for cultured cell, by Thermo Fisher Scientific (1 mentions)
β tubulin, by GeneTex (1 mentions)
Mouse anti ha, by InvivoGen (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Clarity western ecl substrate kit, by Bio-Rad (1 mentions)
Image one software, by Bio-Rad (1 mentions)
Ecl western blotting system, by GE Healthcare (1 mentions)
Anti cox 2, by Santa Cruz Biotechnology (1 mentions)
Hrp anti goat, by Merck Group (1 mentions)
Anti aqp4, by Santa Cruz Biotechnology (1 mentions)
Image4000, by GE Healthcare (1 mentions)
10 protocols using horseradish peroxidase hrp conjugated anti rabbit igg
1
Immunohistochemical Analysis of SOX4 in Oral Lesions
2
Western Blotting and Subcellular Fractionation
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Western blotting was performed as previously described20 (link),27 (link),54 (link),58 ,62 (link). The concentration of the lysates was determined by TaKaRa BCA Protein Assay Kit (T9300A), following the manufacture’s protocol. A3A and A3B were blotted by either a rabbit serum that reacted to both A3A and A3B20 (link). Other antibodies used in this study were as follows: rabbit anti-keratin 10 (K10) (sab4501656, Sigma), rabbit anti-GAPDH (G9545, Sigma), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (GE Healthcare, Little Chalfont, UK), mouse anti-HA (Invivogen, San Diego, California), and anti-mouse IgG–HRP (GE Healthcare).
Subcellular fractionation of W12 cells was performed using a Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher, 89874) following the manufacturer’s instructions. After the removal of nuclei and unlysed cells, supernatants were spun down at 3000 × g for 15 minutes at 4 °C to harvest mitochondria with minimum contamination of lysosomes or peroxisomes. Anti-HSP-60 (GeneTex, Irvine, California, GTX110089) and β-tubulin (GeneTex, GTX101279) antibodies were used to validate the purification of the mitochondrial and cytosolic fractions, respectively.
Subcellular fractionation of W12 cells was performed using a Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher, 89874) following the manufacturer’s instructions. After the removal of nuclei and unlysed cells, supernatants were spun down at 3000 × g for 15 minutes at 4 °C to harvest mitochondria with minimum contamination of lysosomes or peroxisomes. Anti-HSP-60 (GeneTex, Irvine, California, GTX110089) and β-tubulin (GeneTex, GTX101279) antibodies were used to validate the purification of the mitochondrial and cytosolic fractions, respectively.
3
Western Blot Analysis of Lymphocytes and Myeloid Cells
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For western blot analyses, B and T lymphocytes from BM were stained with biotinylated anti-B220 (Clone: RA3.3A1/6.1) and anti-CD3e-APC (Clone: 145-2C11), respectively. Monocytes, neutrophils, macrophages, MP and LSK were stained as described above. Cells were sorted and lysed in Laemmli's sample buffer with 50 mM dithiothreitol, and boiled for 5 min. The equivalent of 105 cells (for the myeloid compartment) or 1.5 × 105 cells (for lymphocytes) was loaded onto each lane of a 7.5% SDS–PAGE gel and after electrophoresis the separated proteins were blotted onto a nitrocellulose membrane (Millipore). Membranes were blocked and incubated with an anti-ESL1-myc rabbit serum (a gift of Dr M.K. Wild; 1:1,000 diluted) and then washed thrice. The primary antibody was detected by using horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (GE Healthcare; 1:5,000 diluted). Membranes were incubated with HRP substrate (Luminata Forte Western HRP substrate, Millipore) and luminescence was recorded on an ImageQuant LAS 4000 mini system (GE Healthcare). To obtain an internal load control, the membranes were then stripped and re-blotted with an anti-β-actin antibody (Sigma; 1:2,500 diluted).
4
Western Blot Analysis of Piscine Reovirus Proteins
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Purified PRV-3 virus particles or blood pellets pooled from three cohabitant fish were mixed with XT buffer and XT reducing agent (Bio-rad), heated for 5 min at 95 °C and loaded onto a 4–12% criterion XT bis–tris gel. Separated proteins were transferred onto a 0.2 µm PVDF membrane using Trans Blot Turbo Transfer system (Bio-rad) and incubated overnight at 4 °C with antiserum against PRV-1 proteins; anti-σ1 (1:1000) [19 (link)], anti-σ3 (1:500) [1 (link)], anti-µ1C (1:500) [19 (link)] and anti-actin (1:500) (Sigma). After washing 4 × 15 min the membranes were incubated with the secondary antibody horse radish peroxidase (HRP)-conjugated anti-rabbit IgG (Amersham, GE Healthcare, Buchinghamshire, UK) (1:20 000). The membranes were washed 4 × 15 min and stained with the Clarity Western ECL Substrate kit (Bio-rad). MagicMark was used as molecular weight ladder (XP Western Protein Standard, Invitrogen). Images were acquired using ChemiDoc XRS+ system and Image one software (Bio-rad).
5
Western Blot Analysis of Protein Markers
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Nitrocellulose membranes were blocked overnight at 4 °C with 2% bovine serum albumin (BSA) in Tris-buffered saline (TBS; in mM 10 Tris, 150 NaCl, pH 7.5, and 0.05% Tween 20®) and then incubated overnight at 4 °C in blocking solution containing the following antibodies: anti-Kir 4.1, anti-AQP-4, anti-COX1, anti-COX2 (diluted 1:1000), and anti-GS (diluted 1:10,000) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-actin (1:2000) (Sigma). Subsequently, the membranes were incubated for 1 h at room temperature in a solution containing horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (diluted 1:10,000), HRP anti-mouse IgG (diluted 1:10,000) (GEHealthcare, Sao Paulo, Brazil), or HRP anti-goat diluted 1:10000 (Sigma). A chemiluminescence signal was detected by luminol substrate reaction (ECL Western Blotting System, GEHealthcare®). Immunoblots were quantified by membrane scanning in an Image4000, GE Healthcare®, and optical densities of proteins studied were determined by ImageJ software (Packard Instrument Company) and the protein/actin ratio calculated.
6
Antibody Validation for Western Blot and IHC
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Antibodies used for western blot analysis were as follows; primary antibodies included guinea-pig anti-human SQSTM1 (1:3000, PROGEN), rabbit anti-human LC3 (1:5000, MBL), rabbit anti-GAPDH (1:5000, Sigma-Aldrich) and mouse monoclonal anti-β-actin (1:10000, Sigma-Aldrich) antibodies. Secondary antibodies included horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5000, GE Healthcare bioscience), HRP-conjugated anti-mouse IgG (1:5000, GE Healthcare bioscience) and HRP-conjugated anti-guinea-pig IgG (1:3000, SantaCruz) antibodies.
Antibodies used for immunohistochemistry were as follows; primary antibodies included rabbit anti-human LC3 (1:1000, MBL), mouse monoclonal anti-mKeima (1:1000, DAKO), isotype control mouse IgG2a (1:100, DAKO) and isotype control rabbit IgG (1:100, DAKO) antibodies. Secondary antibodies included N-Histofine® Simple Stain Mouse MAX PO (Nichirei) and EnVision+ System-HRP Labelled Polymer anti-mouse antibody (DAKO).
Antibodies used for immunohistochemistry were as follows; primary antibodies included rabbit anti-human LC3 (1:1000, MBL), mouse monoclonal anti-mKeima (1:1000, DAKO), isotype control mouse IgG2a (1:100, DAKO) and isotype control rabbit IgG (1:100, DAKO) antibodies. Secondary antibodies included N-Histofine® Simple Stain Mouse MAX PO (Nichirei) and EnVision+ System-HRP Labelled Polymer anti-mouse antibody (DAKO).
7
Cell-based Complement Activation Assay
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The cell-ELISA was performed as described with modifications13 (link). Briefly, Cells were seeded at 10,000 cells/well in 96-well culture plates and incubated overnight at 37 °C in 5% CO2. Cells were then cultured in media containing 10% pooled human serum (Innovative Research, Novi, MI) for 1 h to activate the complement system. Plates were washed with phosphate-buffered saline (PBS) followed by fixing with 3% paraformaldehyde (PFA) for 15 min. The cells were incubated in blocking buffer (5% skim milk in Tris-buffered saline; TBS) for 1 h at 37 °C. A rabbit polyclonal C5b-9 antibody (Abcam, Cambridge, MA) diluted in blocking buffer (1:4,000) was added to the plate, and incubated with the cells for 2 h. The plate was washed three times with TBS/T for 15 min, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (GE Health Care, Buckinghamshire, UK) was added. After incubation at room temperature for 1 h, the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, KPL, Gaithersburg, MD) was used. The absorbance at 450 nm was measured by a microplate reader (Molecular Devices, Silicon Valley, CA).
8
Proteinase K Characterization of Leptospira MPL36
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MPL36 cell surface localization was first assessed by proteinase K treatment as previously described [39 (link)]. Briefly, Manilae WT cells were grown to a density of 5×108 cells/mL and harvested by low-speed centrifugation at 2,000 × g for 10 min at room temperature. The pellet was gently washed with phosphate buffered saline (PBS) containing 5 mM MgCl2, and collected by centrifugation at 2,000 × g for 10 min. After resuspension in PBS-5 mM MgCl2, proteinase K (Sigma-Aldrich) diluted in proteolysis buffer (10 mM Tris-HCl pH8.0, 5 mM CaCl2) was added to the washed leptospires in a final concentration of 25 to 100 μg/mL. Proteolysis buffer without proteinase K was added to the negative control. The reaction was quenched by the addition of phenylmethylsulfonyl fluoride (PMSF). Leptospires were subsequently collected by centrifugation and washed twice with PBS-5 mM MgCl2, and the cells were resuspended in sample buffer for SDS-PAGE. Immunoblot analysis was performed using rabbit antibodies against proteins MPL36, LigA and GroEL at a dilution of 1:1,000. Bound antibodies were detected using horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (GE Lifesciences) at a dilution of 1:100,000. Positive signals were detected by SuperSignal West Pico Kit (Pierce), according to the manufacturer’s instructions, and blots were analyzed using ChemiDoc Imager (Bio-Rad).
9
Western Blot Analysis of Signaling Pathways
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Western blotting was performed according to standard procedures [11] (link), using the following primary antibodies: phospho-Jun (Ser63), phospho-SAPK/JNK (Thr183/Tyr185) Rabbit mAb (98F2), phospho-FoxO1 (Thr24)/FoxO3a (Thr32), FoxO1 (C29H4) Rabbit mAb (all from Cell Signaling Technology, Danvers, MA, USA), GAPDH (Millipore, Billerica, MA, USA). Incubation with secondary antibody [horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or HRP-coupled anti-mouse IgG (GE-Healthcare, Piscataway, USA)] was followed by detection using chemiluminescent HRP substrate. Images were acquired using an ImageQuant LAS 4000 mini system (GE Healthcare Bio-Sciences).
10
Western Blot Analysis of STAT Proteins
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Whole cell lysates were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Immobilon-P; Millipore). The blot was blocked with 5% skim milk or BSA in Tris-buffered saline (TBS; 20 mM Tris-HCl, pH 7.4, 137.5 mM NaCl) containing 0.05% or 0.1% Tween 20 (TBS-T) for 1 h at room temperature. We selected appropriate concentrations of Tween20 and blocking reagent depending on the antibodies used. Then, the membrane was incubated overnight with primary antibodies at 4 °C, washed by TBS-T for 5 min three times, and incubated with appropriate secondary antibodies for 1 h at room temperature. After washing with TBS-T another three times, the membrane was treated with Chemi-Lumi One Super reagent (nakalai tesque) and chemiluminescence was detected using VersaDoc 5000 imager (Bio-Rad). Rabbit polyclonal antibody (pAb) against STAT1 (Santa Cruz Biotechnology), rabbit pAb against STAT2 (Santa Cruz Biotechnology), rabbit pAb against phosphorylated STAT2 (Tyr690) (Cell Signaling Technology), rabbit monoclonal antibody (mAb) against phosphorylated STAT1 (Tyr701) (58D6 ; Cell Signaling Technology), mouse mAb against actin (C-2 ; Santa Cruz Biotechnology) or β-actin (Clone BA3R ; BioVision), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (GE health care), and HRP-conjugated anti-mouse IgG (Jackson Immuno Research) were used.
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