Brain samples were homogenized and sonicated in lysis buffer. Total protein was measured by a NanoDrop apparatus (MaestroGen, Las Vegas, NV, USA). 20 μg protein were size-fractionated using any-kD Mini-Protean TGX gel electrophoresis and then transferred to a nitrocellulose membrane, and membranes were blocked and washed in TBS-T, and incubated overnight with AC, ACC1, ACC2, PC, PCC, MCC, presynaptic synapsin I, postsynaptic PSD95, PSD93, IL-17, IL-6, TNF-α, NFkB, GFAP, GAP43, ICAM-1, BDNF, CXCL 16, OPG and MMP-9 antibodies (Abcam, Cambridge, UK). The following day, membranes were washed and then incubated with horseradish peroxidase-conjugated goat anti-rabbit (Abcam, Cambridge, UK) or goat anti-mouse (Abcam, Cambridge, UK) secondary antibody (diluted 1:1000) for 1 h at room temperature. Protein loading was controlled with an anti-β-actin antibody (Abcam, Cambridge, UK). Protein levels were analyzed densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, MD, USA), corrected with values determined on β-actin blots, and expressed as relative values compared with the control group.
Horseradish peroxidase conjugated goat anti rabbit
Manufactured by Abcam
Sourced in United Kingdom, United States
Horseradish peroxidase-conjugated goat anti-rabbit is a secondary antibody. It is conjugated with the enzyme horseradish peroxidase, which can catalyze a chromogenic reaction for detection purposes.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Brain derived neurotrophic factor (bdnf), by Abcam (1 mentions)
Nf kb, by Abcam (1 mentions)
Cxcl16, by Abcam (1 mentions)
Tnf α, by Abcam (1 mentions)
Il 17, by Abcam (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Icam 1, by Abcam (1 mentions)
Mmp 9, by Abcam (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Gap43, by Abcam (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Abcam (1 mentions)
Iba 1, by Abcam (1 mentions)
Tyrosine hydroxylase th, by Proteintech (1 mentions)
Horseradish peroxidase conjugated goat anti mouse, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
11 protocols using horseradish peroxidase conjugated goat anti rabbit
1
Western Blot Analysis of Brain Proteins
2
Quantification of Amygdalar Proteins in Rats
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The amygdala samples were harvested 24 h after final behavioral test. The amygdala of rats was ultrasonically disrupted in a cold radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology Co., Haimen, Jiangsu, China) with protease inhibitors (PMSF, Beyotime Biotechnology Co., Haimen, Jiangsu, China) followed by centrifugation at 12,000 × g for 20 min. Then, the total protein concentration of the supernatant was quantified by a BCA kit (Beyotime Biotechnology Co., Haimen, Jiangsu, China). Western blotting was performed as described previously (Hiroi et al., 1997 (link)). Primary antibodies used were tyrosine hydroxylase (TH, rabbit polyclonal, 1:2,500; Proteintech, Chicago, IL, United States); glial fibrillary acid protein (GFAP, rabbit polyclonal, 1:3,000; Abcam, MA, United States); Iba1 (rabbit monoclonal, 1:1,000; Abcam); DRD1 (rabbit monoclonal, 1:2,000; Abcam); DRD2 (rabbit polyclonal, 1:1,000; Abcam, MA, United States); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (rabbit monoclonal, 1:5,000; Abcam, MA, United States). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit (1:5,000; Abcam, MA, United States) and horseradish peroxidase-conjugated goat anti-mouse (1:5,000; Abcam, MA, United States).
3
Western Blot Analysis of EP1-4 Receptors in PMN- and M-MDSCs
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Proteins from isolated PMN- and M-MDSCs were extracted. In short, cells were lysed with NP-40 lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Trist-HCl, protease inhibitors (Carl Roth)) for 30 minutes during continuous agitation and spun down at 16000 g for 20 minutes, after which the supernatant was collected. The cell lysates were separated by 10% SDS-PAGE gels in Laemmli loading buffer, and transferred to polyvinylidene fluoride (PVDF) membranes (Invitrogen, Carlsbad, CA, USA). After blocking with 5% skim milk/PBS/0.1% Tween (Carl Roth), blots were probed with primary antibodies for the EP1-4 receptors (Alomone Labs, Jerusalem, Israel, 1:250) and GAPDH (Abcam, Cambridge, UK, 1:2500). Membranes were then incubated with the appropriate secondary horseradish peroxidase-conjugated goat anti-rabbit (Abcam, 1:1000) for 1 hour at room temperature.
4
Immunohistochemical Analysis of TIA1 Expression
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Following peroxidase blocking for 30 min and 5% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) blocking for 2 h at room temperature, frozen sections (20 µm) of the brain were incubated with primary antibody (cat. no. ab140595; rabbit anti-TIA1 antibody 1:1,000; Abcam, Cambridge, MA, USA) overnight. Then these sections were incubated with secondary antibody (cat. no. ab6721; horseradish peroxidase-conjugated goat anti-rabbit; 1:5,000; Abcam) for 2 h at room temperature. The expression of TIA1 was detected using a Streptavidin Peroxidase Immunohistochemical staining kit (Tiangen Biotech Co., Ltd., Beijing, China) with DAB staining. The ratio of positive cells was calculated using Image Pro Plus software (version 6.0; Media Cybernetics, Inc.). The histopathological observations were documented by light microscopy (Leica DMI400; Leica Microsystems) at 400× magnification and images were captured. Each experiment was replicated 3 times and the data are presented as the mean ± SEM.
5
Western Blot Immunodetection of PKN1
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The purified
recombinant PKN1
proteins were separated by 10–14% SDS-PAGE and then transferred
to nitrocellulose membranes for immunostaining. The membrane was probed
with either polyclonal anti-PKN1-(539–556) or rabbit monoclonal
anti-PKN1 (Abcam) antibodies at room temperature, before being washed
with 1 × Tris buffer saline-Tween-20 (1× TBS-T) wash buffer,
followed by addition of secondary antibody (horseradish peroxidase-conjugated
goat antirabbit; Silenus or Abcam). The immunoreactive bands were
detected by enhanced chemiluminescence (ECL) (Bio-Rad Western ECL
substrate or Kodak BioMax).
recombinant PKN1
proteins were separated by 10–14% SDS-PAGE and then transferred
to nitrocellulose membranes for immunostaining. The membrane was probed
with either polyclonal anti-PKN1-(539–556) or rabbit monoclonal
anti-PKN1 (Abcam) antibodies at room temperature, before being washed
with 1 × Tris buffer saline-Tween-20 (1× TBS-T) wash buffer,
followed by addition of secondary antibody (horseradish peroxidase-conjugated
goat antirabbit; Silenus or Abcam). The immunoreactive bands were
detected by enhanced chemiluminescence (ECL) (Bio-Rad Western ECL
substrate or Kodak BioMax).
6
Quantitative Western Blot Analysis
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Protein samples were quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer’s protocols. Protein samples were heated with SDS sample buffer (0.25 M Tris–HCl, pH 6.8, 0.5 M DTT, 10% SDS, 50% glycerol and 0.5% bromophenol blue) at 95 °C for 5 min, a total of 30 μg were loaded per lane and then separated on 15% SDS-PAGE and electroblotted onto polyvinylidene fluoride membranes (GE10600023, GE Healthcare Life Sciences, Logan, UT, USA). Subsequently, the membranes were incubated with one of the primary rabbit polyclonal antibodies at a dilution of 1:1,000 (Abcam) or anti-β-actin rabbit monoclonal antibody at a dilution of 1:5,000 (Abcam) overnight at 4 ℃. Immunoreactive bands were detected by incubation with horseradish peroxidase-conjugated goat anti-rabbit (Abcam) at a dilution of 1:5,000 for 2 h at room temperature. Detection by chemiluminescence was performed using a Pierce enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
7
Visualizing Pancreatic Islet Vasculature
8
Protein Expression Analysis by Western Blot
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Following transfection, the cells were washed three times with PBS and lysed with radioimmunoprecipitation assay lysis buffer to obtain the total protein. Protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Total proteins (50 µg) were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes, as previously described (20 (link)). The membranes were incubated with blocking buffer (5% skimmed milk) for 1.5 h at room temperature and then incubated with the following primary antibodies: anti-CDK6 (1:500, ab79454; Abcam, Cambridge, MA, USA) and anti-GAPDH antibody (1:500; SAB4300645-100UG; Sigma-Aldrich, Merck KGaA) at 4°C overnight. Following incubation with the primary antibody, blots were washed with TBS-0.1% Tween and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:2,000, ab6721) or rabbit anti-mouse IgG (1:2,000, ab6709) (both from Abcam) secondary antibodies at 37°C for 2 h. Band intensity was quantified using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA, USA). Experiments were performed in triplicate.
9
Exosomal Protein Characterization by Western Blot
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We separated 20 μg of exosomal proteins by 10% SDS-PAGE for 90 min at 120 V. The proteins were transferred onto polyvinylidene fluoride membranes (Merck Millipore). Next, we incubated the membranes in blocking solution (5% milk in PBS containing 1% Tween-20) for 1 h at 20°C and then incubated with rabbit monoclonal antibodies against mouse CD9 (1:1,000) (SBI, USA) and CD63 (1:1,000) (SBI) to detect EVs markers. We used monoclonal rabbit anti-mouse β-actin (1:1,000) (Bioss, China) as an internal control. The membranes were washed and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:3,000) (Abcam, United Kingdom) for 1 h at room temperature. Finally, we visualized the protein bands using the Piece Fast Western Blot Kit and ECL Substrate (Thermo Scientific), and analyzed the bands using Image Lab software (Bio-Rad, USA).
10
Immunoblotting Analysis of Apoptosis-related Proteins
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Cells or kidney tissue samples were lysed with lysis buffer containing protease inhibitors. Equal amounts of protein were dissolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes. After being blocked with 5% nonfat milk, the membranes were immunoblotted with primary antibodies against B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-Associated X (Bax), cleaved cysteinyl aspartate specific proteinase (caspase)-1, Apoptosis associated speck-like protein containing caspase recruitment domain (ASC), Recombinant Human NACHT, LRR and PYD domains-containing protein 3 (NLRP3), DDX5, and GAPDH at a dilution of 1:1000 overnight at 4℃. Following washing, the membranes were exposed to horseradish peroxidase-conjugated goat anti-rabbit (1:5000, Abcam, Cambridge, UK) secondary antibodies, and signals were detected with the enhanced chemiluminescence detection system. All antibodies mentioned above were purchased from Abcam (Cambridge).
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