Antibodies used for Western blotting and immunofluorescence were as follows: anti-α-SMA (ab5694; Abcam, Cambridge, England), anti-collagen Type I (600-401-103-0.1; Rockland, Inc., PA, USA), anti-fibrinogen (ab23750, Abcam), anti-Periostin (ab14041, Abcam), anti-Smad2 (#3103S; Cell Signaling, Beverly, MA, USA), anti-phosphorylated Smad2 (#3101S; Cell Signaling), anti-Stat3 (#9139S; Cell Signaling), and anti-phosphorylated Stat3 (#9145S, Cell Signaling). Antibodies for the immunohistochemistry experiments were as follows: anti-E-cadherin mAb (M3612; Dako, Carpinteria, CA, USA), anti-α-SMA (ab5694; Abcam), and anti-Ki67 (M7240; Dako). Transforming growth factor (TGF)-β1 was purchased from R&D Systems (240-B; Minneapolis, MN, USA). For in vitro studies, PFD was provided by Shionogi & Co., Ltd. (Osaka, Japan) and dissolved in DMSO to a concentration of 50 μg/mL. For in vivo studies, Pirespa tablets were purchased from Shionogi & Co. and dissolved in DMSO, using one-fifth of the final volume of the total solvent, after crushing using a micro smash (TOMY SEIKO CO., LTD, Tokyo, Japan). Sterile normal saline at four-fifths of the final total volume was then added to bring PFD to a final concentration of 40 mg/mL.
Micro smash
Manufactured by TOMY
Sourced in Japan
The Micro Smash is a compact laboratory equipment designed for the crushing and grinding of small samples. It features a sturdy construction and a simple, intuitive interface for efficient sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Pe 2400, by PerkinElmer (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Ab14041, by Abcam (1 mentions)
Ab23750, by Abcam (1 mentions)
Transforming growth factor β1, by R&D Systems (1 mentions)
Anti phosphorylated stat3, by Cell Signaling Technology (1 mentions)
Anti ki67, by Agilent Technologies (1 mentions)
Anti phosphorylated smad2, by Cell Signaling Technology (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Anti α sma, by Abcam (1 mentions)
Anti smad2, by Cell Signaling Technology (1 mentions)
Ab5694, by Abcam (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Estradiol elisa kit, by Cayman Chemical (1 mentions)
3 mm zirconia beads, by Biospec (1 mentions)
Ultrafree mc plhcc 250 pk for metabolome analysis, by Human Metabolome Technologies (1 mentions)
Agilent capillary electrophoresis system, by Agilent Technologies (1 mentions)
17 protocols using micro smash
1
Antibody-based Analysis of Fibrosis Markers
2
Quantitative Real-Time PCR of Tissue Samples
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Intestinal mucosa or liver biopsy tissues were homogenized using Micro Smash (TOMY, Tokyo, Japan), and total RNA was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany). The cDNA was synthesized from RNA using a High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA) after quantification and purity assessment using a Nanodrop (Thermo Fisher). Quantitative real-time PCR (qRT-PCR) was conducted using a StepOnePlus real-time PCR system (Applied Biosystems) with a TaqMan probe (Table 4
3
Peripheral Estradiol Quantification
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Peripheral tissues (the caudal halves of the bodies) were collected from cck2rb +/+ and cck2rb -/-females at 2-4.5 h after the onset of the light period, frozen at -80 °C, and homogenized with Micro Smash (Tomy Seiko Co. Ltd., Tokyo, Japan). Tissue lipids were extracted with diethyl ether. Tissue levels of E2 were determined using the Estradiol ELISA Kit (Cayman Chemical Company, Ann Arbor, MI). Peripheral levels of E2 were expressed as picograms per mg tissue weight.
4
Metabolomic Analysis of Fecal Samples
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Fecal metabolites were extracted from fresh thawed fecal samples (about 10 mg) suspended in 400 μL of 50% methanol in Millli-Q water supplemented with the internal standards (20 μM each of methionine sulfone and D-camphor-10-sulfonic acid (CSA)). The mixture was combined with two 3-mm zirconia beads (BioSpec Products, Bartlesville, OK, USA) and about 100 mg of 0.1-mm zirconia/silica beads (BioSpec Products, Bartlesville, OK, USA) and subjected to 3 min of vigorous shaking using a Micro Smash (TOMY, Nerima, Tokyo, Japan). The suspension was centrifuged at 4600× g for 15 min at room temperature, and the resulting supernatant was transferred to a 5-kDa-cutoff filter column (Ultrafree MC-PLHCC 250/pk for Metabolome Analysis, Human Metabolome Technologies, Tsuruoka, Yamagata, Japan). The flow-through was dried under vacuum and the residue then was dissolved in 40 μL of Milli-Q water containing reference compounds (200 μM each of 3-aminopyrrolidine and trimesate). The levels of extracted metabolites were measured in both positive and negative modes by CE-TOFMS as previously described [74 (link)]. All CE-TOFMS experiments were performed using an Agilent capillary electrophoresis system (Agilent Technologies, Santa Clara, CA, USA).
5
Quantification of Immune Markers in Mouse Brain
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Mice were euthanized under isoflurane anesthesia and perfused with chilled PBS. Brains were removed and immersed in a solution containing RNAlater (Thermo Fisher Scientific, Tokyo, Japan) and incubated overnight at 4 °C. Immersed brains were homogenized using Micro Smash (Tomy Seiko, Tokyo, Japan). Total RNA was extracted from tissue homogenates using the RNeasy mini kit (Qiagen, Germantown, MD, USA), and cDNA was synthesized from 1 µg of RNA using the TaqMan Fast Cells-to-CT kit (Thermo Fisher Scientific). The concentration of RNA in each sample was determined using NanoDrop (Thermo Fisher Scientific). The TaqMan real-time polymerase chain reaction assays using probes and primers for Tlr2, Tnf and 18S rRNA (Applied Biosystems, Waltham, CA, USA) were performed using the QuantStudio 7 Flex system (Applied Biosystems). For an endogenous reference gene, 18S rRNA was used. Fold changes were determined using the ΔΔCT method.
6
Quantifying Lung Myeloperoxidase Activity
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MPO activity was measured using a myeloperoxidase fluorometric detection kit (Enzo Life Science) and an fMax fluorescence microplate reader (Molecular Devices) with fMax-pro software according to the manufacturers' protocols. Briefly, lung tissue specimens were homogenized with MicroSmash (TOMY) in 1 mL of assay buffer containing hexadecyltrimethylammonium bromide. The protein concentration in the tissue homogenates was measured using the Micro BCA Protein Assay (Pierce), and 2.5 μg of the lysate was placed in each assay well. All samples were assayed twice in duplicate. The data are presented as units of MPO enzymatic activity per 100 μg of tissue protein.
7
Isolation of Matsutake Mycorrhizal Fungi
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Matsutake ectomycorrhizal root tips were picked from shiro soil and from the seedlings of P. densiflora under a stereomicroscope. The root tips, 3-5 mm length, were rinsed five times with sterile water followed by stirring in a beaker first in 0.05% Tween 20 for 2 min and then in 0.001% Tween 20 for 30 min. Twenty mycorrhizal root tips per shiro sample were collected into a 2 ml tube with Zirconia beads, 2.0 mm (TOMY, Japan). Warm HNC-medium (200 μl) containing yeast extract (60 g), CaCl 2 (0.5 g), SDS (5 g) in 1 L water (Nonomura and Hayakawa 1988) was added. The root tissues were disrupted using a Micro Smash ™ (TOMY, MS-100, Japan) with 20 s pulses at 2000 rpm, repeated three times, then incubated at 42 °C for 30 min. The tubes were centrifuged at 8000 rpm for 2 min. The supernatant was looped by a glass loop onto ISP2 agar medium (Shirling and Gottlieb 1966) (link) surface, following a distinctive zig-zag pattern to ensure obtaining a single colony. The cultures were incubated at 28 °C in the dark. Later, a single colony was transferred onto a fresh ISP2 medium in the same conditions.
8
GST Pull-down Assay for Protein Interactions
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200 ml of yeast cells were grown on YPD or selective media at 30 °C to A600 nm = 1.5. Normalized cells were harvested, washed and transferred into 2 ml screw-capped tubes. Cells were lysed with glass beads (Sigma) and beaten with Micro Smash (TOMY) for 4 times (1 minute per time). Lysed cells were centrifuged at 10,000 g for 10 minutes at 4 °C. Supernatant was transferred to new microcentrifuge tubes. 30 µl of GST beads (GE Health Lifesciences) were washed with lysis buffer (50 mM Tris-HCL, 150 mM KCL, 1% NP-40) and resuspended in 100 µl of lysis buffer. Cell extracts were each mixed with 40 µl GST beads in 100 µl of lysis buffer and protease inhibitor cocktail (Calbiochem, 1:1000). The mixture was rotated at 4 °C for 2 hours. After incubation, the mixture was washed with lysis buffer 5 times. The mixture was added with 5X SDS loading dye (5% β-Mercaptoethanol, 0.02% Bromophenol Blue, 30% Glycerol, 10% SDS, 250 mM PH6.8 Tris-CL) and heated at 95 °C for 10 minutes.
9
Cecal DNA Extraction Protocol
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Procedures of DNA extraction from cecal contents were conducted according to Matsuki’s method.(20 ) Cecal samples (20 mg) were washed two times by resuspending them in 0.2 ml of PBS and centrifuging each preparation at 14,000 × g to remove possible PCR inhibitors. Following the second centrifugation, the cecal pellets were resuspended in a solution consisting of 0.2 ml of PBS, 250 µl of extraction buffer (200 mM Tris-HCl, 80 mM EDTA. pH 9.0), and 50 µl of 10% sodium dodecyl sulfate. A total of 300 mg of glass beads (diameter 0.1 mm) and 500 µl of buffer-saturated phenol were added to the suspension, and the mixture was vortexed vigorously for 180 s using a MicroSmash (Tomy Seiko Co., Ltd, Tokyo, Japan) at 4,000 rpm. After centrifugation at 14,000 × g for 5 min, 400 µl of the supernatant was collected. Phenol–chloroform–isoamyl alcohol extraction was then performed, and 250 µl of the supernatant was subjected to isopropanol precipitation. Finally, the obtained DNA was dissolved in 1 ml of 10 mM Tris-EDTA buffer, pH 8.0. The DNA solution was adjusted to a final concentration of 10 µg/ml in the same buffer.
10
Soil Carbon Analysis Protocol
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All samples of the O layer and mineral soils were analyzed as follows.
(1) O layer The samples were dried in an oven (70 °C, 48 hours), and their dry weights were measured. We then roughly ground them by using a Wiley mill (W-100S, IRIE SHOKAI Co. Ltd., Japan) and then further ground small amounts using a bead cell disrupter (Micro Smash, TOMY, Japan). Finally, the carbon concentration of each sample was measured using a CHNS/O analyzer (PE 2400, Perkin Elmer Co., Ltd., Japan).
(2) Bulk density of mineral soils After air drying, we classified each sample collected in the 100-mL core into fine soil particles, gravel, and roots by passing them through a 2 mm sieve. Each fine soil sample was dried in an oven (105 °C, 24 hours), and its dry weight was measured. The bulk density was calculated according to the dry weight of soil particles (excluding gravel and roots) divided by the volume (i.e., 100 mL).
(3) Carbon concentration in mineral soils
The mineral soil collected for measuring the carbon concentration was air dried and then passed through a 2 mm sieve followed by a 500 μm sieve. The carbon concentration of each sample was measured using a CHNS/O analyzer (PE 2400).
(1) O layer The samples were dried in an oven (70 °C, 48 hours), and their dry weights were measured. We then roughly ground them by using a Wiley mill (W-100S, IRIE SHOKAI Co. Ltd., Japan) and then further ground small amounts using a bead cell disrupter (Micro Smash, TOMY, Japan). Finally, the carbon concentration of each sample was measured using a CHNS/O analyzer (PE 2400, Perkin Elmer Co., Ltd., Japan).
(2) Bulk density of mineral soils After air drying, we classified each sample collected in the 100-mL core into fine soil particles, gravel, and roots by passing them through a 2 mm sieve. Each fine soil sample was dried in an oven (105 °C, 24 hours), and its dry weight was measured. The bulk density was calculated according to the dry weight of soil particles (excluding gravel and roots) divided by the volume (i.e., 100 mL).
(3) Carbon concentration in mineral soils
The mineral soil collected for measuring the carbon concentration was air dried and then passed through a 2 mm sieve followed by a 500 μm sieve. The carbon concentration of each sample was measured using a CHNS/O analyzer (PE 2400).
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