Tissue collected were fixed for 2 h at room temperature in 4% PFA. This was followed with 3x wash of 1x PBS (Amresco, Solon, Ohio, USA). Tissues were then infused under a sucrose gradient for cryo-protection before cryo-embedding in Tissue-Tek® O.C.T. Compound (Sakura Finetek, Torrance, California, USA). Prior to antigen staining, cryo-sections were permeabilized in 0.5% TritonX-100 (Chem Supply, Gillman, South Australia) before blocking with 20% normal goat serum. In this study, primary antibodies used includes rat anti-mouse CD31 (1:100), rabbit anti-αSMA (1:200), rabbit anti-FSP1 (1:200), rabbit anti-SLUG (1:200), rabbit anti-SOX9 (1:500), rabbit anti-ERG (1:500) and mouse anti-SOX9 (1:200). Primary antigen staining was conducted overnight at 4 °C. Sections were then subjected to 3 × 5 min washes in 1x PBS + 0.1% Tween-20 (Amresco, Solon, Ohio, USA). Secondary antibodies conjugated with Alexa-fluor 568 or 647 (Invitrogen, Carlsbad, CA, USA) were used for fluorescence detection. Sections were incubated with secondary antibodies for 40 min at room temperature. Nuclear staining was revealed in specimens mounted with ProLong® Gold mounting media containing DAPI (Invitrogen, Carlsbad, CA, USA).
Phosphate buffered saline (pbs)
Manufactured by Avantor
Sourced in United States, Poland, United Kingdom, Germany, France, Italy, Austria, Spain
Phosphate-buffered saline (PBS) is a commonly used buffer solution in biological and biochemical research. PBS is a balanced salt solution that maintains a physiological pH and osmolarity, suitable for a variety of laboratory applications. It is a versatile and widely used product for various experimental procedures.
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Lab products found in correlation
Fetal bovine serum (fbs), by Avantor (8 mentions)
Trypsin edta, by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Diva software, by BD (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Ethanol, by Avantor (4 mentions)
Rpmi 1640, by Avantor (4 mentions)
L glutamine, by Avantor (4 mentions)
Hepes, by Avantor (4 mentions)
Percoll, by GE Healthcare (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Hydrochloric acid (hcl), by Avantor (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Acetonitrile, by Merck Group (4 mentions)
Sodium hydroxide (naoh), by Merck Group (4 mentions)
Dimethyl sulfoxide (dmso), by Avantor (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Neutral buffered formalin, by Avantor (3 mentions)
Tween 80, by Merck Group (3 mentions)
Methanol, by Avantor (3 mentions)
142 protocols using phosphate buffered saline (pbs)
1
Multimarker Analysis of Tissue Sections
2
Osteoblast Mineralization Quantification
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After 7 days of incubation, discs with osteoblasts were washed 3 times with PBS (VWR®, Radnor, PA, USA) to remove non-adherent cells. Cells were fixed using 2.5% glutaraldehyde (VWR®, Radnor, PA, USA) for 1 h and then the discs were washed with PBS. Cell culture mineralization was evaluated using the OsteoImageTM Mineralization Assay (PA-1503, Lonza, Morristown, NJ, USA). This assay consists of the fluorescent staining of extracellular mineral content deposited by cells, specifically hydroxyapatite. Mineralization-stained images were obtained at 492/529 nm excitation/emission wavelengths using a Leica TCS SP5 confocal microscope (Leica Microsystems, Deerfield, IL, USA) coupled to LAS-AF LITE v2.0 software (Leica Microsystems, Deerfield, IL, USA).
3
Quantifying Cell Growth Strategies
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To test the growth of cells using the ON, OFF, and QS strategies, primary cultures were diluted 1000X in 5 mL fresh media. For ON cells, public good production was induced by adding 3 μg/mL of 3-oxo-C6-acylhomoserinelactone (AHL, Adipogen) as in (Wellington and Greenberg, 2019 ). Upon reaching OD600nm of 0.3, cells were washed three times 1X PBS (VWR, life science) and resuspended in 1 mL 1X PBS. Cells at 100X dilution were then inoculated into 5 mL fresh M9 media supplemented with 0.0125% glucose and 0.05% starch (Sigma- Aldrich). For the ON cells, 3 μg/mL of AHL was used to induce public good production. The cell density within triplicate cultures was monitored every hour for 18 h by counting colony forming units on LB agar with 50 μg/mL kanamycin.
4
Experimental Protocol for Mouse EAE Model
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The reagents used in this study were purchased as described: THC and CBD from Cayman Chemical (Michigan, USA), myelin oligodendrocyte glycoprotein (MOG35−55) peptide H-MEVGWYRSPFSRVVHLYRNGK-OH (PolyPeptide Laboratories, San Diego, CA, USA). Mycobacterium tuberculosis (strain H37Ra) (BD, Franklin Lakes, NJ, USA), complete Freund's adjuvant (Fisher, Hampton, NH, USA), Pertussis toxin (List Biological Laboratories, Campbell, CA, USA), Percoll, GE Healthcare Life Sciences (Pittsburgh, PA, USA); Neural Tissue Dissociation Kit (P) (Miltenyi Biotech, Auburn, CA, USA), RBC lysis buffer (Sigma-Aldrich, St. Louis, MO, USA), RPMI 1640, l-glutamine, HEPES, phosphate-buffered saline, and fetal bovine serum (VWR, West Chester, PA, USA), ELISA Max Kits IL-10, IL-17A, IFN-γ, IL-6, IL-1β, TNF-α, and TGF-β and FITC Annexin V/-PI apoptosis kit (Biolegend, San Diego, CA). EasySep PE selection kit (Stemcell Technologies, Cambridge, MA, USA), Propidium Iodide (PI)/RNase Staining Solution (Cell Signaling Technology, Danvers, MA, USA), miRNeasy Mini Kit, miScript II RT Kit and miRNAs primers (Qiagen, Valencia, CA), mRNAs primers (Integrated DNA technologies, Coralville, IA, USA) and SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA).
5
Histomorphometry of Avian Intestinal Villi
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Mid-jejunal segments were collected from 1 bird per replicate with a median BW, flushed with ice-cold 10% phosphate-buffered saline (VWR International, Radnor, PA) and fixed in 10% neutral buffered formalin (VWR International, Radnor, PA) for approximately 30 d. Subsequently, the samples were dehydrated with ethanol (VWR International, Radnor, PA), cleared with Sub-X (Polysciences, Inc., Warrington, PA) and placed in paraffin (Polyfin paraffin, Sigma Polysciences, St. Louis, MO). The segments (5 μm) were stained with hematoxylin and eosin at the Purdue Histology and Phenotyping Laboratory (Purdue University, West Lafayette, IN). The villus height and crypt depth were measured from 5 complete, vertically oriented villi per slide, and subsequently, the villus height-to-crypt depth ratio was calculated. All measurements were performed under a binocular light microscope (National Optical and Scientific Instruments, Inc., Schertz, TX).
6
Isolation and Preservation of Lymphatic Vessels
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Tissue specimens were transported directly from surgical theatre to the laboratory in ice-cold sterile Phosphate Buffered Saline (VWR, UK). LVs were identified and isolated along with a small amount of surrounding tissue under a dissection microscope within a sterile laminar flow cabinet (Fig 1A–1C ). Each vessel was divided into two sections. One half of each vessel was further cleaned of surrounding fatty tissue, placed in RNA stabilising solution (RNAlater, ThermoFisher, UK), frozen on dry ice and stored at -20°C. The other half was fixed in 4% paraformaldehyde (VWR, UK) for 45 minutes, followed by submersion in 15% sucrose for 3 hours, then 30% sucrose for 6–12 hours. This was followed by embedding in OCT (Scigen, UK) using dry ice and isopropyl alcohol (ThermoFisher, UK) and stored at -80°C.
7
Carvedilol Formulation Development
8
Extraction and Dilution of Plant Leaf Extracts
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Leaf tissue of N. benthamiana or E. sonchifolia was collected 7 days post infection, transferred to an extraction bag (Bioreba, Switzerland) and mixed with 5 ml of phosphate buffered saline (VWR, West Chester, PA), hand homogenized using a hand-held tissue homogenizer (Agdia, Elkhart, IN) and kept at room temperature for the remainder of the experiment. Serial dilutions of this extract were made, when necessary, by mixing 20 µl of extract with 180 µl of nuclease-free water and repeating this process in series for up to 20 times.
9
Lentiviral Transfection and GFP Protein Analysis
10
Preparation and Characterization of Lipid-Based Nanoparticles
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1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, MW 790), cholesterol (CHOL, ovine wool, MW 387), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (18:0 PEG2000 PE, MW 2805), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000 amine, MW 2791) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). GA (MW 170), holo-Tf human (MW 80,000), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, MW 192), thioflavin T (ThT, MW 319), chloroform (MW 119), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, MW 168) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Human amyloid-β peptide with 42 amino acid residues (Aβ1-42, MW 4514) was acquired from GenScript Biotech (Piscataway, NJ, USA). Uranyl acetate dihydrate (MW 424) was purchased from Electron Microscopy Sciences (Hatfield, PA, USA). Diethyl ether (MW 74) was obtained from José Manuel Gomes dos Santos, Lda (Odivelas, Portugal), while methanol (MW 32) and ethanol absolute (MW 46) were bought from VWR International (Radnor, PA, USA). VWR International also provided phosphate-buffered saline (PBS) tablets (pH 7.4, 10 mM phosphate buffer, 2.7 mM potassium chloride, and 137 mM sodium chloride). PBS was prepared using ultrapure water (Milli-Q Academic, Millipore, Burlington, MA, USA).
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