It was used to detect the expression of microglia activated marker IBA1, M1 phenotypic marker iNOS protein, and M2 phenotypic marker Arg-1 protein. Briefly, microglia were lysed in RIPA strong lysis buffer on ice and then centrifuged at 4°C and 12000 rpm for 10 min to extract nucleoprotein, and quantitative analysis of protein content was performed by BCA (Beyotime, Shanghai, China) method. The protein components of the sample were separated on SDS-PAGE gels (BIO-RAD, Hercules, CA, USA) and transferred to the PVDF membrane (Biosharp, Suzhou, China). After blocking with 5% skimmed milk (BIO-RAD, Hercules, CA, USA), the membranes were incubated with the primary antibodies against GAPDH, IBA1, iNOS, and Arg-1 overnight at 4°C. After the primary antibody incubation is completed, the PVDF membranes were incubated with HRP conjugated IgG antibodies for 1 h at room temperature and analyzed by an image with a Chemiscope imaging system (CLiNX, Shanghai, China). The intensities of target protein bands were quantified and normalized by GAPDH using ImageJ (NIH, Bethesda, MD).
Skimmed milk
Manufactured by Bio-Rad
Sourced in United States
Skimmed milk is a laboratory reagent used as a blocking agent in various immunoassays and Western blot techniques. It is effective in reducing non-specific binding of antibodies to membranes or solid support surfaces, thereby improving the specificity of the target analyte detection.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Chemidoc system, by Bio-Rad (2 mentions)
Horse radish peroxidase conjugated species specific secondary antibodies, by Jackson ImmunoResearch (2 mentions)
Enhanced chemiluminescence reaction, by Merck Group (2 mentions)
Trans blot turbo transfer kit, by Bio-Rad (2 mentions)
Protease inhibitor cocktail tablet, by Cell Signaling Technology (2 mentions)
Bca method, by Thermo Fisher Scientific (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Sds page, by Bio-Rad (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Pvdf membrane, by Biosharp (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Chemiscope imaging system, by Clinx (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Rabbit anti lc3 antibody, by Novus Biologicals (1 mentions)
Rabbit anti p62 antibody, by Abcam (1 mentions)
Rabbit anti beclin 1 antibody, by Abcam (1 mentions)
19 protocols using skimmed milk
1
Quantifying Microglia Activation Markers
2
Western Blot Analysis of Autophagy and Apoptosis Markers in Osteoblasts
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Osteoblasts were lysed in RIPA lysis buffer with protease inhibitors (Beyotime, Shanghai, China). Proteins were collected after centrifugation. The protein concentrations were determined by BCA Protein Assay Kit (Beyotime, China). The protein samples were subjected to SDS-PAGE (8–12%), and then transferred onto PVDF membranes (Millipore, Germany). After blocking with 5% skimmed milk (Bio-Rad, USA) for at least 1 h, the membranes were incubated with primary antibodies overnight at 4°C, including rabbit anti-LC3 antibody (1 : 1000, Novus), rabbit anti-Bnip3 antibody (1 : 2000, Abcam), rabbit anti-P62 antibody (1 : 1000, Abcam), rabbit anti-Beclin1antibody (1 : 1000, Abcam), mouse anti-caspase 3 antibody (1 : 500, Santa Cruz), rabbit anti-cleaved caspase 3 antibody (1 : 1000, CST), rabbit anti-Bcl-2 antibody (1 : 1000, CST), rabbit anti-Bax antibody (1 : 1000, CST) and mouse anti-β-actin antibody (1 : 8000, Tianjin Sungene Biotech). Depending on the origin of the primary antibodies, the secondary antibody (1 : 7000 – 1 : 10000) was added and incubated for 1 h at room temperature. The intensities of bands were quantified with Fusion software (VILBER LOURMAT, Germany). All the results were normalized to β-actin.
3
Western Blot Analysis of Skeletal Muscle
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The skeletal muscle tissue (EDL) was homogenized using RIPA lysis buffer (Thermo, 89,900, IL, USA) supplemented with Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (Thermo, 1,861,280, IL, USA). The resulting supernatant was collected, and the total protein content was measured using the Rapid Gold BCA Protein Assay Kit (Thermo Scientific, A53226, IL, USA) according to the manufacturer’s instructions. Proteins (25 µg) were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels ranging from 8 to 12%. The separated proteins were then transferred onto polyvinylidene difluoride membranes (Bio-Rad, 1,620,177, CA, USA) and blocked with 5% skimmed milk (Bio-Rad, 1,706,404, CA, USA) for 2 h. Afterward, the membranes were washed three times with TBST and incubated overnight at 4 °C with primary antibodies. The primary antibodies used in this study included AKT, p-AKT (Ser473), mTOR, p70S6K, p-p70S6K, and p-FOXO3A (all from Cell Signaling Technology, MA, USA; 1:1000), as well as FOXO3A, FOXO1, NF-κB, and MuRF1 (all from Proteintech, Wuhan, China; 1:1000). A secondary antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Cell Signaling Technology, Danvers, MA, USA; 1:2000), was applied. The protein bands were visualized using the ChemiDoc system (Bio-Rad, California, USA) and quantified using Image J software.
4
Western Blot Analysis of Protein Extracts
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Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EDTA plus 10 µg/ mL aprotinin, 10 µg/ mL leupeptin, 1 mM PMSF, 1 mM Na2VO4) at +4 °C according to standard procedures. Twenty-five total protein extracts were resolved on 12% SDS/PAGE and electrically transferred onto poly(vinylidene difluoride) membranes (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked with TBS-T (20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.02% Tween-20) containing 5% skimmed milk (Bio-Rad Laboratories) for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies diluted in TBS-T containing 5% milk: rabbit polyclonal anti-Tom20 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) and mouse monoclonal anti-GAPDH (1:5000; Sigma-Aldrich). After thorough washing in TBS-T, immunocomplexes were revealed with horseradish-peroxidase-conjugated species-specific secondary antibodies (Jackson Laboratory, Bar Harbor, ME, USA) followed by an enhanced chemiluminescence reaction (Millipore Corporation, Darmstadt, Germany). Reactive bands were detected by the ChemiDoc MP system (Bio-Rad Laboratories) and signal quantification was performed using the IMAGE LAB software 5.0 (Bio-Rad Laboratories).
5
Analyzing Intestinal Organoid Responses
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Bat and human intestinal organoids were lysed and subjected to total RNA extraction using the RNeasy kit, followed by reverse transcription using the PrimeScript RT-PCR kit (Takara, PR014B) and an oligo(dT) primer. The resultant cDNAs were used to measure mRNA expression levels of cellular genes using LightCycler 480 SYBR Green I Master Mix (Roche) and an LC480 thermocycler (Roche). Data were analyzed by the delta-delta Ct method. qPCR primers were listed in Supplementary Table 2 .
For Western blot, human intestinal organoids were lysed in RIPA buffer supplemented with protease inhibitors (Roche). The lysates were separated in 12% SDS-PAGE and then transferred to a 0.22 µm PVDF membrane (Bio-Rad). After overnight blocking with 5% skimmed milk (Bio-Rad), the membrane was incubated with an anti-ISG15 antibody (Thermo Fisher Scientific, MA5-15029) for 2 h at room temperature, followed by incubation with an HRP-conjugated secondary antibody and detection with immobilon crescendo western HRP substrate (Millipore). Cell-free media were harvested from treated and mock-treated human intestinal organoids for measuring the amount of human IFNL1 and IFNL3 using ELISA kits (R&D Systems, DY7246, D28B00).
For Western blot, human intestinal organoids were lysed in RIPA buffer supplemented with protease inhibitors (Roche). The lysates were separated in 12% SDS-PAGE and then transferred to a 0.22 µm PVDF membrane (Bio-Rad). After overnight blocking with 5% skimmed milk (Bio-Rad), the membrane was incubated with an anti-ISG15 antibody (Thermo Fisher Scientific, MA5-15029) for 2 h at room temperature, followed by incubation with an HRP-conjugated secondary antibody and detection with immobilon crescendo western HRP substrate (Millipore). Cell-free media were harvested from treated and mock-treated human intestinal organoids for measuring the amount of human IFNL1 and IFNL3 using ELISA kits (R&D Systems, DY7246, D28B00).
6
Western Blot Analysis of ICAM-1 and CD36
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Cells were lysed in boiled Sample Buffer 1x (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 100 mM DTT). In total, 30 μg of total protein extracts were resolved on 8% SDS-PAGE and electrically transferred onto poly(vinylidene difluoride) membranes (PVDF, Bio-Rad Laboratories, Hercules, CA, USA) membranes. Membranes were blocked with TBS-T (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.02% Tween-20) containing 5% skimmed milk (Bio-Rad), for 1 h at room temperature, and then incubated overnight at 4°C with primary antibodies diluted in TBS-T containing 5% milk. The following antibodies were used: mouse monoclonal anti-ICAM-1 (Santa Cruz Biotechnology, Dallas, TX, USA; working dilution 1:500), rabbit polyclonal anti-CD36 (Santa Cruz Biotechnology, working dilution 1:500), mouse monoclonal anti-α-tubulin (Sigma-Aldrich, working dilution 1:10,000). After extensive washing, immunecomplexes were detected with horse-radish peroxidase conjugated species-specific secondary antibodies (Jackson Laboratory, Bar Harbor, ME, USA) followed by enhanced chemiluminescence reaction (Millipore Corporation, Billerica, MA, USA). Proteins detected by immunoblotting were quantified by densitometry (ChemiDoc imaging system, BioRad) and normalized as a function of α-tubulin with Image-Lab 5.0 software (Bio-Rad).
7
Protein Extraction and Western Blot Analysis
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The cells were cultivated in serum-free DMEM in six-well plates until they reached 80% confluency. The medium was removed, and the cells were washed twice with PBS. A lysis buffer (M-PER® Mammalian Protein Extraction Reagent; Pierce Biotechnology, Rockford, IL, USA) was then added to the cells, following which they were scraped from the plate and centrifuged at 14,000 × g at 4°C for 10 min. The supernatant was then heated for 5 min in Laemmli sample buffer containing dithiothreitol (50 mM). Equivalent quantities of the lysed and heated proteins (30 μg/well) were electrophoresed on polyacrylamide gels (10%; Bio-Rad, Shanghai, P.R. China), following which the isolated proteins were transferred to a polyvinylidene difluoride membrane (GE Healthcare, Pittsburgh, PA, USA). The membrane was blocked with skimmed milk (5%; Bio-Rad) for 1 h at room temperature, and then incubated overnight with the primary antibody at 4°C. Determination of concentrations was conducted as instructed. The membrane was then incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, and the proteins were finally examined using enhanced chemiluminescence kits (GE Healthcare).
8
Western Blot Protein Analysis Protocol
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Cell and tissue extracts were prepared using cell lysis buffer (Cell Signaling Technology; #9803S) with protease inhibitor cocktail tablets (Cell Signaling Technology; #5871). The BCA method (Thermo Fisher Scientific) was used for protein quantification. Protein (20 μg) was separated using SDS-PAGE (Bio-Rad). Trans-blot Turbo Transfer kit (Bio-Rad) was used for semi-dry transfer. After transfer, membranes were blocked using 5% skimmed milk (Bio-Rad) in TBST (TBS buffer containing 0.5% Tween-20) for 1 hour, followed by overnight incubation with primary antibodies (in 5% milk and dilution as per the manufacturer's instructions) at 4°C. On day −2, the membrane was washed 4 times with TBST buffer and incubated with the respective HRP-conjugated secondary antibody (1:5,000) for 1 hour. Further, membranes were washed four times with TBST buffer and developed using ClarityTM Western ECL Substrate (Bio-Rad; #1705061). Primary antibodies used in this study are described in Supplementary Table S2.
9
Western Blot Protocol for Protein Detection
10
Kir4.1-Peptide ELISA Assay Protocol
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