Sesn1+/- and Sesn1-/- mice were vaccinated with FLUAD as above described. One week later, mice were sacrificed and blood samples were immediately collected by cardiac bleeding. Levels of circulating immunoglobulins (IgGs) were analyzed using a serum dilution of 1:200 in 0.1% non-fat dry milk 0.5% Tween-20/PBS, as described 33 (link). Samples were incubated on Nunc Maxisorp plates pre-coated with FLUAD overnight (1:40; for 18 hours at 4 °C), or PBS as background control. Plates were washed 3 times with 0.5% Tween-20/PBS and blocked with 200 μl of 4% non-fat dry milk (GE-Healthcare). Antigen-specific serum antibodies were detected using horseradish peroxidase (HRP) conjugated antibodies (anti-mouse IgG, Sigma Aldrich) at 1:3000 dilution in 0.1% non-fat dry milk 0.5% Tween-20/PBS, at room temperature. Substrate activity was detected using 100 μL of tetramethylbenzidine (TMB) substrate (BD Biosciences) and stopped using 50 μL 2N H2SO4 per well. Circulating vaccine-speciific IgG levels were determined by ELISA (absorbance emission at 450 nm) of triplicate wells ± s.e.m.
Non fat dry milk
Manufactured by GE Healthcare
Sourced in United Kingdom, Italy, Sweden
Non-fat dry milk is a powdered form of milk that has had its fat content removed. It is a common ingredient used in various laboratory applications where a concentrated source of milk protein and carbohydrates is required, without the presence of fat.
Automatically generated - may contain errors
Lab products found in correlation
Tetramethylbenzidine tmb substrate, by BD (2 mentions)
Non fat dry milk, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Nonfat dry milk, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal α sma antibody, by Abcam (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Signalboost immunoreaction enhancer kit, by Merck Group (1 mentions)
Anti β actin monoclonal antibody, by Merck Group (1 mentions)
Protein sds page loading buffer, by Takara Bio (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ecl chemiluminescent reagent, by GE Healthcare (1 mentions)
Hyperfilm mp, by GE Healthcare (1 mentions)
Ecl detection kit, by GE Healthcare (1 mentions)
Synaptophysin, by Merck Group (1 mentions)
Syntaxin, by Abcam (1 mentions)
Biii tubulin, by Merck Group (1 mentions)
Mem media, by Corning (1 mentions)
Bradford assay, by Merck Group (1 mentions)
7 protocols using non fat dry milk
1
Sestrins Regulate Influenza-Specific Antibody Response
2
Sestrins Regulate Influenza-Specific Antibody Response
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Sesn1+/- and Sesn1-/- mice were vaccinated with FLUAD as above described. One week later, mice were sacrificed and blood samples were immediately collected by cardiac bleeding. Levels of circulating immunoglobulins (IgGs) were analyzed using a serum dilution of 1:200 in 0.1% non-fat dry milk 0.5% Tween-20/PBS, as described 33 (link). Samples were incubated on Nunc Maxisorp plates pre-coated with FLUAD overnight (1:40; for 18 hours at 4 °C), or PBS as background control. Plates were washed 3 times with 0.5% Tween-20/PBS and blocked with 200 μl of 4% non-fat dry milk (GE-Healthcare). Antigen-specific serum antibodies were detected using horseradish peroxidase (HRP) conjugated antibodies (anti-mouse IgG, Sigma Aldrich) at 1:3000 dilution in 0.1% non-fat dry milk 0.5% Tween-20/PBS, at room temperature. Substrate activity was detected using 100 μL of tetramethylbenzidine (TMB) substrate (BD Biosciences) and stopped using 50 μL 2N H2SO4 per well. Circulating vaccine-speciific IgG levels were determined by ELISA (absorbance emission at 450 nm) of triplicate wells ± s.e.m.
3
Western Blot Analysis of α-SMA and GAPDH
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The cells were washed with cold PBS, harvested with a cell lifter, centrifuged and lysed with RIPA lysis buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% Na deoxycholate and 0.1% SDS) supplemented with a complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis IN), homogenized with a pellet pestle motor (Kimble/Kontes, Vineland, NJ) and subsequently centrifuged at 13,000 x g for 15 min at 4°C. For Western blots either aliquots of culture media containing the proteins secreted by the cultured cells or aliquots of the cell lysates containing cellular proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA). Blots were blocked for 1 h in Tris-buffered saline(TBS)-Tween (10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 0.1% Tween 20) containing 5% nonfat dry milk (BioRad, Hercules, CA). The membranes were then incubated overnight at 4°C with mouse monoclonal α-SMA antibody (Abcam, 1:200), and GAPDH polyclonal rabbit antibody (Abcam, 1:2000) in a 5% nonfat dry milk/TBS-Tween solution. Membranes were then washed with TBS-Tween, and incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, UK) diluted 3000-fold in 5% nonfat dry milk/TBS-Tween. The signals were quantified using NIH Image J software.
4
Western Blot Analysis of FGFR Proteins
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Protein extracts from cell lysates were obtained with RIPA buffer mixed protease inhibitor cocktail (Thermo scientific). Protein extractions were boiled in 4× Protein SDS-PAGE Loading Buffer (Takara) for 5 min and resolved on a SDS-PAGE, and then transferred onto 0.45-μm NC membrane (GE Whatman). Membranes with total FGFR proteins were blocked by 5% (w/v) non-fat dry milk (GE Healthcare), while those with phosphorylated FGFR1 protein were treated with 5% (w/v) bovine serum albumin (BSA) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies including anti-FGFR1 polyclonal antibody (1:500 dilution, Cell Signaling Technology), anti-phosphorylated FGFR1 polyclonal antibody (1:1000 dilution, Lianke Biotechnology Ltd.), anti-FGFR4 monoclonal antibody (1:1000 dilution, Abcam; Epitomics), and anti-β-actin monoclonal antibody (1:5000 dilution, Sigma-Aldrich). After three washes with TBS containing 0.1% (v/v) Triton X-100 (TBST), they were incubated with goat anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution, Cell Signaling Technology) for 1 h at room temperature. Final detection of protein was performed using the Signal Boost Immunoreaction Enhancer Kit (Merck Millipore). Protein levels were quantified using Quantity One software (Bio-Rad).
5
Western Blot Analysis of MMP-9 and TIMP-1
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Samples of conditioned media containing 40 µg of total proteins, were mixed with a reducing sample buffer and subjected to 10% SDS-PAGE. Separated proteins were then trans-blotted onto polyvinilidene difluoride (PVDF) transfer membranes. The non-specific protein binding site on membranes was blocked by incubation in a solution containing 5% non-fat dry milk (Biorad, Milan, Italy) in TBS 0.1% Tween 20 (TBS-T) for 1 h, and then incubated overnight at 4 °C with the primary antibodies for human MMP-9 and TIMP-1 diluted in 1% non-fat dry milk in TBS-T. Membranes were then washed in TBS-T, incubated for 1 h with secondary HRP-conjugated antibody diluted in blocking solution, and the immunoreactive bands were detected by inducing a chemiluminescence reaction through the ECL chemiluminescent reagent (GE Healthcare, Little Chalfont, England). Quantitative analysis of immunoreactive spots was performed by using ImageJ software [36 ].
6
Western Blotting for CYP26B1 Quantification
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Details on the method used for western blotting are provided in our previous report [7 (link)]. Mouse antihuman CYP26B1 monoclonal antibodies (Abnova, Taipei City, Taiwan) were used as the first antibody at a 1 : 1000 dilution, and a horseradish-peroxidase conjugated antimouse antibody (Cell signaling Co.) was used as the second antibody at a 1 : 1000 dilution. Both antibodies were dissolved in 3% nonfat dry milk (GE Healthcare, Uppsala, Sweden) in 0.05% TBST (8 g NaCl, 0.2 g KCl, 3 g Tris, 0.5% Tween 20, dd H2O to 1 L, and pH 7.4). Peroxidase activity was detected using an ECL detection kit (GE Healthcare) and recorded using the HyperFilm TMMP (GE Healthcare).
7
Hippocampal Slices Immunoblotting Analysis
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The hippocampal slices were obtained from 8-to 12-week-old C57BL/6 male mice killed by decapitation. The temporal lobes were cut into 350-mm-thick slices with vibratome. The hippocampal slices were then cultured for immunoblotting in MEM media (Corning) and treated with 5-mM GlcNAc and 150-mM UDP-GlcNAc or 50-mg/mL Ab 25e35 for 1 week. Slices were then homogenized in RIPA buffer containing protease inhibitors (Roche Diagnostics). After 2 hours at 4 C, homogenates were centrifuged at 4000 rpm to discard cellular debris. Protein content was determined by Bradford Assay (Sigma-Aldrich). Protein lysates were diluted in Laemli buffer, boiled at 90 C for 5 minutes, and then resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto the nitrocellulose membrane (Bio-Rad), which were blocked with Tris-buffered saline and 0.1% Tween 20% and 10% nonfat dry milk (GE-Healthcare). Membranes were then incubated at 4 C overnight with the following primary antibodies: syntaxin (Abcam), synaptophysin (Millipore), bIII tubulin, and actin (Sigma-Aldrich). Appropriate secondary IgG HRP-conjugated (GE Healthcare) was added for 1 hour, and chemiluminescent detection was performed with ECL Plus advanced (Amersham and GE Healthcare). Quantitative analysis of the signal obtained was performed by Image J software (National Institutes of Health).
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