Hippocampal tissue samples were homogenized in a lysis buffer (Kurabo, Osaka, Japan). The homogenates were centrifuged, and the supernatants were obtained. Western blotting was performed as previously described [24 (link)]. The membranes were incubated at room temperature for 1 h with primary antibodies against brain and muscle arnt-like 1 (Bmal1) (1:1000; Cell Signaling Technology, Danvers, MA, USA), Cryptochrome 1 (Cry1) (1:1000; Proteintech Group, Rosemont, IL, USA), Cry2 (1:1000; Proteintech Group, Rosemont, IL, USA), ionized calcium-binding adapter protein 1 (Iba1; marker of microglia (1:1000; Wako, Osaka, Japan), CC-Chemokine receptor 7 (CCR7) (1:1000; Abcam; Cambridge, MA, USA), and β-actin (1:5000; Sigma-Aldrich, St. Louis, MO, USA) as loading controls. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Novex, Frederick, MD, USA). Immune complexes were detected using ImmunoStar Zeta reagent (Wako, Osaka, Japan), and images were captured using Multi Gauge Software ver. 3.0 (Fujifilm, Greenwood, SC, USA).
Multi gauge software ver 3
Manufactured by Fujifilm
Sourced in Japan
Multi Gauge software ver. 3.0 is a measurement and analysis tool for industrial applications. The software provides advanced features for data acquisition, processing, and reporting.
Automatically generated - may contain errors
Lab products found in correlation
Lysis buffer, by Kurabo (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Bmal1, by Cell Signaling Technology (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
Cd163, by Thermo Fisher Scientific (1 mentions)
Las 4000 luminescent image analyzer, by Fujifilm (1 mentions)
Imagequant las 4000, by Fujifilm (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Las 3000, by Fujifilm (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
5 protocols using multi gauge software ver 3
1
Hippocampal Protein Expression Analysis
2
Macrophage Subtype Analysis by Western Blot
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The dorsal skin samples were homogenized in lysis buffer (Kurabo, Osaka, Japan). Following this, the homogenates were centrifuged at 8000× g for 10 min, and the resultant supernatants were collected. Western blotting was performed as previously described [23 (link)]. Briefly, the membranes were incubated with primary antibodies against F4/80 (marker of macrophage; 1:1000; ThermoFisher Scientific, Waltham, MA, USA), chemokine receptor 7 (CCR7: marker of M1 type macrophage; 1:1000; Abcam, Cambridge, MA, USA), CD163 (marker of M2 type macrophage; 1:1000; ThermoFisher Scientific), or β-actin as a loading control (1:5000; Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h. The membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibody (Novex, Frederick, MD, USA). Immune complexes were detected using ImmunoStar Zeta reagent (Wako, Osaka, Japan), and images were acquired using Multi Gauge software ver. 3.0 (Fujifilm, Greenwood, SC, USA).
3
TGFβ1-Induced Protein Expression in NCI-H292 Cells
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NCI-H292 cells (5 × 105 cells/well) were seeded in 6-well plates. The cells were incubated for 12 h in GM, and the medium was subsequently changed to serum-reduced GM medium (0.1% FBS). After 16 h, the cells were treated with the respective concentration of TGFβ1 for 30 min. Proteins were prepared and loaded as described elsewhere (Lee et al., 2014 (link)). At least 30 µg of protein from the whole cell lysate was used per sample for western blot analysis. The band intensity was visualized using a LAS-4000 luminescent image analyzer (Fujifilm, Japan) and quantified by densitometry (Fuji Multi Gauge software ver. 3.0).
4
Gelatin Zymography for MMP-9 Activity
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We assessed the gelatinolytic activity of MMP-9 by performing a gelatin zymography assay, as described in [24 (link)]. First, 5 μL of culture supernatants were electrophoresed in 10% polyacrylamide gels containing 1% sodium dodecyl sulfate (SDS) and 0.1% gelatin. The gels were washed twice in 2.5% Triton X-100 for 30 min and incubated overnight at 37 °C in incubation buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM CaCl2, and 1 nM ZnCl2. The gels were then stained in 0.25% Coomassie brilliant blue R-250 and destained using 35% ethanol and 10% acetic acid solution. Gelatinolytic activity was quantified by scanning densitometry with an ImageQuant LAS-4000 biomolecular imaging system (Fujifilm, Tokyo, Japan) and analyzed by Multi Gauge software, ver. 3.0 (Fujifilm).
5
Protein Expression Analysis by Western Blot
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For Western blotting, muscle samples were incubated ex vivo in the same manner as mentioned above except for the addition of the radioisotopes. Then, protein was extracted from the samples using lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, 1 mM NaF, pH 8.0) with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) and 0.1 mM PMSF. Ten micrograms of total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Merck Millipore), blocked with Blocking One (Nacalai Tesque, Kyoto, Japan), and washed with TBST (TBS with 0.1% Tween20). The blots were incubated with the primary antibodies shown in Supplementary Table S1 , and reaction products were visualized using a horseradish peroxidase-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA, United States) and enhanced chemiluminescence (Thermo Fisher Scientific). Each protein was detected by LAS 3000 (Fujifilm, Tokyo, Japan). Band densities were measured with Multi Gauge software Ver3.0 (Fujifilm).
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