Ex vivo 2,3,5-triphenyl tetrazolium chloride (TTC, Sigma, USA) staining was performed to assay mitochondrial electron transport activity of adipose tissues [25 (link)]. Briefly, each tissue was minced to approximately 30 mg and incubated in PBS with 2% TTC for 15 min at 37 °C. Sequentially, tissues were fixed with 10% formalin for 30 min at room temperature. For the formazan product extraction, each tissue samples were separately transferred into 95% ethanol and incubated at 4 °C overnight. The absorbance of extracted solutions was measured using MultiSkan Go spectrophotometry (Thermo Fisher, USA) at 485 nm and the values were normalized with tissue weights.
Multiskan go spectrophotometry
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MultiSkan Go is a compact spectrophotometer designed for absorbance measurements. It can be used to quantify various biomolecules and perform a range of common laboratory assays. The instrument provides accurate and reproducible results across a broad wavelength range.
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Lab products found in correlation
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Toptaq dna polymerase kit, by Transgene (1 mentions)
Abi 2720 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Agencourt ampure xp magnetic beads, by Beckman Coulter (1 mentions)
Fusion solo chemiluminescence imaging system, by Vilber (1 mentions)
Sigmafast protease inhibitor, by Merck Group (1 mentions)
Pro prep, by iNtRON Biotechnology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
4 protocols using multiskan go spectrophotometry
1
Quantifying Mitochondrial Activity in Adipose Tissue
2
Evaluation of Blood-Brain Barrier Permeability
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To detect the BBB permeability 48 hours after SE, mice were first administered 2% Evans blue (EB, 4 mL/kg; Sigma-Aldrich) through the tail vein 4 hours before sacrifice. After perfusing the mice with normal saline, the hippocampal tissue was then isolated, weighed, and homogenized in cold phosphate buffered saline. After adding 50% trichloroacetic acid (Aladdin Reagents Co., Ltd., Shanghai, China) to precipitate the protein, we performed centrifugation at 12,000 × g, 4°C, for 30 minutes to obtain the supernatant. The dye concentration in the supernatant was determined using Thermo Fisher Scientific Multiskan GO spectrophotometry at 620 nm. To further compare EB leakage between groups, we analyzed the coronal brain slices under a fluorescence microscope.
3
Profiling Gut Microbiome From Fecal Samples
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Fecal samples were collected in sterile plastic cups, frozen, and stored at -80°C within 1 h until further processing. Fecal microbial DNA was extracted using a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). PCR amplification was carried out using an ABI 2720 Thermal Cycler (Thermo Fisher Scientific, USA). We used Multiskan™ GO spectrophotometry (Thermo Fisher Scientific, USA) to quantify bacterial genomic DNA as the template for amplification of the V3-V4 hypervariable region of the 16S rRNA gene in three replicate reactions with forward (Illumina adapter sequence 1 + 5’-CCTACGGGNBGCASCAG) and reverse (Illumina adapter sequence 2 + 5’-GGACTACNVGGGTWTCTAAT) primers. Replicate PCR products were pooled and purified with Agencourt AMPure XP magnetic beads (Beckman Coulter, USA). A TopTaq DNA Polymerase kit (Transgen, China) was used. The purity and concentration of sample DNA were assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, USA). Paired-end sequencing was performed by Treatgut Biotech Co., Ltd. with a HiSeq 2500 (Illumina, San Diego, CA, USA) with PE 250 bp reagents.
4
Protein Extraction and Western Blot Analysis
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Protein from adipose tissues was extracted by protein extraction solution PRO-PREP (iNtRON Biotechnology, Seongnam-si, Korea), with PhosSTOP phosphate inhibitor (Roche, Basel, Switzerland) and SIGMAFAST Protease Inhibitor (Sigma, Livonia, MI, USA). The concentration of protein was determined by MultiSkan GO spectrophotometry (Thermo Fisher, Waltham, MA, USA) with Pierce BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA) at 562 nm. Proteins were separated by size on the SDS-PAGE gel (8% or 12%) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h in blocking solution (5% bovine serum albumin or powdered skim milk in TBST) and then incubated with primary antibodies overnight. After washing the membranes, they were incubated with secondary antibodies (HRP-conjugated anti-mouse, anti-rabbit secondary antibodies, Thermo Fisher, Waltham, MA, USA) for 1 h. The protein bands were visualized using Fusion Solo chemiluminescence imaging system (Vilber Lourmat, Collégien, France) and EvolutionCapt program (ver. 17.03). The quantification of immunoblots was using ImageJ (National Institutes of Health, Bethesda, MD, USA). The primary antibodies were listed in Table S2 .
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