Homogenizer

Excised tissues sliced into ∼2-mm² pieces and preserved in RNA Later (QIAGEN) were mechanically dissociated in 500 μL Qiazol lysis reagent (QIAGEN) using a homogenizer (OMNI International), and total RNA was extracted using the RNeasy Lipid Tissue Mini Kit (QIAGEN). Alternatively, hippocampus and SVZ regions were dissected and dissociated using the adult brain dissociation kit from Miltenyi Biotec and the gentleMACS Octo Dissociator with heaters. Cells were then sorted on a FACSDiva 8.0.1 (BD Biosciences) directly into lysis solution (QIAzol) using the following antibodies as reported (Daynac et al., 2013 (link)): fluorescein isothiocyanate mouse LEX/CD15 (BD Biosciences, cat. no. 560127, clone MC480), PE rat CD24 (BD Biosciences, cat. no. 553262, clone M1/69), Alexa 647-conjugated EGF (Molecular Probes E35351). One μg or less of RNA was then reverse-transcribed using the QuantiTect Reverse Transcription Kit (QIAGEN). Quantitative differences in gene expression were determined by real-time qPCR using SYBR Green PCR Master Mix (Bio-Rad) and a spectrofluorometric thermal cycler (Mx3000P from Stratagene). Values are presented as the ratio of target mRNA to 18S rRNA obtained using the relative standard curve method of calculation. Primers sequences were described previously (Le et al., 2010 (link)).

One lobe in each rat (five rats in each group) was selected after the radiologist reviewed the correlated chest CT. The radiologist chose lobes with lesions similar to those in the other lobes. Lung tissues were lysed in a T-PER™ Tissue Protein Extraction Reagent (Thermo Scientific, Rockford, IL, USA) using a homogenizer (OMNI International, Waterbury, CT, USA). Equal amounts of protein extracts (20 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using a natural gel and then transferred onto a polyvinylidene fluoride membrane (Atto, Tokyo, Japan). After blocking with 5% non-fat skim milk for one hour, the membrane was incubated overnight with rabbit anti-collagen type I (1:1,000, Abcam, Cambridge, UK), rabbit anti-fibronectin (1:1,000, Abcam), and mouse β-actin (1:5,000, Santa Cruz, CA, USA). Afterward, an appropriate horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG antibody (Cell Signaling Technology, Danvers, MA, USA) was used to bind to the primary antibodies. Protein band imaging was done using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories).

The cultivated liquid culture with mycelial mass was homogenized at maximum speed with a homogenizer (Omni International, Kennesaw, GA, USA) for 1 min and then centrifuged at 12,000× g for 20 min. After the supernatant was treated with ammonium sulfate powder (30% saturation) for 6 h, the solution was centrifuged to remove the protein precipitate and then ammonium sulfate was added in the supernatant to 80% saturation and maintained overnight to induce the protein precipitate including β-glucosidase and followed by centrifuging (12,000× g) for 20 min. After the precipitate was dissolved in a small volume of 10 mM sodium acetate buffer (pH 4.5) and purified by overnight dialysis with 10 mM acetate buffer (pH 4.5), the supernatant obtained was lyophilized for 4 days. All experiments were carried out at 4 °C unless otherwise mentioned.

The emulsifying agents (PsPC and whey protein concentrate) were prepared at 1% w/v in deionized water and solubilized with constant stirring. Then the sample was allowed to hydrate overnight at 4 °C. Afterwards, 0.05% sodium azide was mixed to prevent microbial growth. Different amounts of canola oil 0.1, 1, 5, 10, and 20 g were added, to obtain a variety of mass fractions (ϕ = 0.002, 0.02, 0.1, 0.2, and 0.4). The emulsions were mixed in a homogenizer (OMNI International GLH, Georgia, United States) at an initial speed of 1000 rpm for 2 min and subsequently at 3000 rpm for 3 min. All emulsions were made in triplicate and stored at 25 ± 2 °C for 18 days, and every three days, all the following analyzes were made. Whey protein concentrate (WPC) was used as a control [42 (link),43 (link)].

For RNA isolation, ~ 10 zebrafish embryos were collected in fenozol (A&A Biotechnology), frozen and stored at -80 °C. Then, the embryos were homogenized using a homogenizer (OMNI International), and total RNA was extracted using the phenol–chloroform method. cDNA was synthesized using M-MLV reverse transcriptase (Promega), and quantitative real-time PCR was performed with SYBR Green Master Mix (A&A Biotechnology) and a QuantStudio3 thermocycler (Thermo Fisher Scientific). Rps11, eef1, acbt2 and rpl13a were used as a reference genes^{49 (link),50 (link)} The primer sequences are listed in Supplementary Table S1. To validate specificity of the qRT-PCR reaction, melt curve analysis was performed at the end of each assay. Agarose gel electrophoresis was performed to ensure presence of a single product of predicted length at the end of the qRT-PCR reaction (Supplementary Fig. S3d).