Parrafin embedded lung tissue sections were stained for fibronectin (Sigma, F3648), vWF (Abcam, ab6994) and reactive oxygen species (ROS, Abcam, ab5512) with negative control isotypes used on serial sections (Dako). Sections were imaged using an Olympus BX60 microscope and densitometry was analysed with ImageJ software.
F3648
Manufactured by Abcam
F3648 is a laboratory equipment product. It is designed to perform a specific function in a laboratory setting.
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Lab products found in correlation
Bx60 microscope, by Olympus (1 mentions)
Ab6994, by Abcam (1 mentions)
Ab5512, by Abcam (1 mentions)
Ab34710, by Abcam (1 mentions)
Ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ab8448, by Abcam (1 mentions)
Mouse anti β actin, by Cell Signaling Technology (1 mentions)
Phosstop phosphatase inhibitor, by Roche (1 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)
Irdye 680rd goat anti mouse igg, by LI COR (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti fibronectin, by Merck Group (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Intercept tbs blocking buffer, by LI COR (1 mentions)
Pierce bca assay, by Thermo Fisher Scientific (1 mentions)
Anti mlkl, by Abcam (1 mentions)
Mab374, by Merck Group (1 mentions)
Enhanced chemiluminescence substrate kit, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using f3648
1
Immunohistochemical Analysis of Lung Tissue
2
Protein Extraction and Western Blot Analysis
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Cell pellets were resuspended in 500 μl RIPA buffer (10X RIPA buffer with protease inhibitors). Lysates were incubated on a shaker for 1 h at 4°C, after which they were centrifuged for 30 min at 19,467 × g to remove cell debris. Supernatant protein concentration was assessed using the Bradford assay, 20 μg total protein was resolved using a 10% SDS-PAGE gel at 180 V for 50 min, and then transferred onto PVDF membranes at 22 V overnight. Membranes were blocked in 5% dried milk in phosphate buffered saline (PBS). Primary and HRP-conjugated secondary antibodies were resuspended at the appropriate concentrations in 5% dried milk in PBS. Western blot analysis was performed with rabbit anti-Fibronectin (1:50,000, Sigma, F3648), rabbit anti-Collagen I (1:500, Abcam, ab34710), rabbit anti-dentin matrix protein 1 and rabbit anti-dentin phosphohoryn [1:1000 (Hao et al., 2009 (link))], rabbit anti-osteopontin (1:1000, Abcam, ab8448), and mouse anti-β-actin antibodies (1:1000, Cell Signaling Technologies, cst3700S); and developed using Pierce enhanced chemiluminescence (ECL) Plus western blotting substrate (ThermoFisher, Cat. No. 32106).
3
Western Blot Analysis of BEC Proteins
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Confluent BECs in 6-well plates were exposed to in vitro grown VTP (3 × 107 cells/well) or equally diluted IEC for 5 h with 3 biological replicates per treatment condition. Cells were lysed for 30 min on ice with gentle agitation in RIPA lysis buffer containing EDTA-free Protease inhibitor cocktail set 3 (Calbiochem, San Diego, CA) and PhosSTOP phosphatase inhibitor (Roche, Mississauga, ON) according to manufacturer’s instructions. Protein concentration was measured using a Pierce BCA assay (Thermo Fisher Scientific). For western blotting, the 3 biological replicates of BEC lysate from each treatment group (VTP- or IEC-exposed) were pooled, and 13.5 μg pooled whole cell lysate per treatment group was subjected to SDS-PAGE using Bolt 12% acrylamide gels (Thermo Fisher Scientific) and transferred to PVDF membrane (Millipore) via wet-transfer at 400 mA for 2 h, and blocked with Intercept TBS blocking buffer (Licor, Lincoln, NE). Anti-fibronectin (1:1,000) (Sigma-Aldrich, F-3648), anti-MLKL (1:1,000) (Abcam, pS358 EPR9514), and anti-GAPDH (1:1,000) (Cell Signaling Technology, D4C6R) were used as primary antibodies, while the secondary antibodies used were goat anti-rabbit IgG IRDye 800CW (1:20,000) and goat anti-mouse IgG IRDye 680RD (1:20,000) (Licor). Detection and analysis were completed on a Licor Odyssey CLx using Licor Image Studio version 5.2.
4
Western Blot Analysis of ECM Proteins
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Samples were lysed in radioimmunoprecipitation assay buffer [50 mM tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, 0.15% Na-Deoxycholate, 1% NP-40, and 2 mM EDTA (pH 8.0)] containing cOmplete protease inhibitors (Roche, no. 11697498001). Protein content was quantified by standard bicinchoninic acid (BCA) assay using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, no. 23227). Equal protein amounts (10 μg/sample) were run on gradient 4 to 12% bis-tris protein gels (Thermo Fisher Scientific, no. NP0329BOX) and transferred to nitrocellulose membranes (Sigma-Aldrich, no. GE10600016). Membranes were blocked in 5% milk solution for 2 hours at RT and probed overnight at 4°C with primary antibodies directed against fibronectin (Sigma-Aldrich, F3648), collagen IV (Abcam, no. ab6586), and GAPDH (Millipore, no. mab374) in 1:1000 dilution. After three washes in 1× Tris-buffered saline with 0.1% Tween® 20 detergent (TBST) buffer (10 min each), membranes were incubated with peroxidase-labeled anti-rabbit (Cytiva, no. NA934V5) or anti-mouse (Abcam, no. ab205719) secondary antibody (1:10,000) for an hour at RT. After subsequent washes in 1× TBST buffer, enhanced chemiluminescence substrate kit (Thermo Fisher Scientific, no. 32132) was used for band detection according to the manufacturer’s instructions. Membranes were imaged on the ChemiDoc MP imaging system (Bio-Rad Laboratories Inc.).
5
Tissue-Specific SAgs Detection in Exosomes
6
Immunohistochemical Staining Protocol for Fibronectin
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Standard procedures for IHC were utilized as previously described (33 (link)). The OCT was removed via two washes in ddH2O for 2 minutes each before dried by subsequent washing in 70% ethanol for 2 minutes, 100% ethanol for 2 minutes, and then left to dry at RT for 10 minutes. Sections were then rinsed with 1X PBS for 5 minutes before incubated in 0.1% Triton X (Sigma-Aldrich REFX100) at RT for 15 minutes to permeabilize cell membranes. Slides were then blocked in Superblock Blocking Buffer in PBS (REF37580, Thermo Fisher Scientific) for 60 minutes at RT before incubated at 4°C overnight with FN (Sigma-Aldrich Corp., F3648) at 1:250, and FN-EDA (Abcam, ab6328) at 1:100 dilution. Primary antibody was washed off with four rinses in 1X PBS for 5 minutes each before slides were incubated in the appropriate secondary antibody for 2 hours at RT; Alexa Fluor 488 donkey anti-rabbit IgG (A21206, Invitrogen – Thermo Fisher Scientific) at 1:200 dilution and Alexa Fluor 594 donkey anti-mouse IgG (A21203, Invitrogen – Thermo Fisher Scientific) at 1:200 dilution. Slides were washed 5 times in 1X PBS for 5 minutes each and mounted with Prolong Gold mounting medium containing DAPI (Invitrogen-Molecular Probes). Image acquisition was performed using Zeiss Axio Imager Z2 microscope. All images were taken at 40X magnification, scale bar represents 20μm.
7
Western Blot Analysis of Protein Expression
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Cells were lysed using lysis buffer containing 50 mM Tris (pH 7.5),150 mM NaCl, 10% glycerol, and 0.5% Nonidet P-40 that was supplemented with protease inhibitors. Western blotting was performed under standard conditions. Blots were repeated and quantified 2–3 times per experiment. Representative images of those repeated experiments are shown. Antibodies used were polyclonal WWOX,60 (link) monoclonal GAPDH (Calbiochem, 6C5, CB1001), polyclonal anti-Fibronectin for immunoblotting (Sigma Aldrich, F3648), polyclonal anti-Fibronectin for immunohistochemistry (Abcam, Ab2413), anti-Myc (Santa Cruz, Sc-40), anti-Flag (Sigma Aldrich, F1804) and polyclonal Smad2/3 (Santa Cruz, sc-8332).
8
Immunofluorescence Analysis of Extracellular Matrix Proteins
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MeWo parental and MeWo SOX10 knockout cells (5 × 105), CRT cells (3 × 105), and A375 parental cells (1.25 × 105) were plated on coverslips coated with 0.2% bovine gelatin and treated with ascorbic acid (50 µg/ml) every 48 h for 6 days. Cells were processed as previously described68 (link),70 (link). Briefly, samples were washed with DPBS + and fixed/permeabilized for 20 min using 4% paraformaldehyde with 5% sucrose and 0.1% Triton X-100 added. Samples were then washed twice with DPBS- with 0.05% Tween-20 and blocked for 1 h at room temperature in Odyssey Blocking Buffer containing 1% donkey serum. Samples were stained with FN1 (Sigma, #F3648, 1:200) and collagen IV (Abcam, ab86042, 1:75) antibodies diluted in Odyssey Blocking Buffer for 1 h at room temperature. After three washes with DPBS- containing 0.05% Tween-20, samples were incubated with secondary antibodies (AlexaFluor-488 Invitrogen A-11034 1:1000 and AlexaFluor-594 Invitrogen A-11032 1:1000 or AlexaFluor-647 Invitrogen A-21236 1:1000) for 30 min at room temperature. Nuclei were stained with DAPI (Abcam, ab228549, 1:1000). After washing three times in DPBS- with 0.05% Tween-20, coverslips were mounted and pictures were taken with A1R Nikon confocal Ti-Eclipse inverted microscope (Nikon, Melville, NY) using NIS-Elements software.
9
Multimodal Cardiac Tissue Analysis
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Hearts were collected and fixed in 2% paraformaldehyde, embedded in Tissue-Tek OCT compound (Sakura Finetek Europe B.V.) and cryosectioned to 16 μm thickness. Immunofluorescence staining was performed as previously described [24 (link)]. To avoid cross-reactivity of antibodies in the multiple immunofluorescence procedure, sequential staining was performed in this study. Antibodies against Col XIIa were incubated after the immunolabeling with the other antibodies.
The following primary antibodies were used: mouse anti-tropomyosin at 1:100 (DSHB, CH1), rabbit anti-Aldh1a2 at 1:200 (GeneTex, GTX124302), rabbit, anti-αSMA at 1:2000 (GeneTex, GTX100034), mouse anti-Vimentin 40E-C at 1:50 (DSHB, 40E-C), rabbit anti-Tenascin at 1:500 (USBioLogical, T2550-23), rabbit anti-Fibronectin at 1:200 (Sigma-Aldrich, F3648), rabbit anti-p-Smad3 at 1:400 (Abcam, ab52903), mouse anti-embCMHC at 1:50 (N2.261; developed by H.M. Blau, obtained from Developmental Studies Hybridoma Bank), rabbit anti-Col1a1 at 1:200 (GeneTex, GTX124368) and guinea pig anti-ColXIIa1 at 1:1000 (Bader et al, 2009). Secondary antibodies (Jackson Immunoresearch) were used at 1:500 and Phalloidin 390 (ATTO, AD 390–81) was used at 1:500.
The following primary antibodies were used: mouse anti-tropomyosin at 1:100 (DSHB, CH1), rabbit anti-Aldh1a2 at 1:200 (GeneTex, GTX124302), rabbit, anti-αSMA at 1:2000 (GeneTex, GTX100034), mouse anti-Vimentin 40E-C at 1:50 (DSHB, 40E-C), rabbit anti-Tenascin at 1:500 (USBioLogical, T2550-23), rabbit anti-Fibronectin at 1:200 (Sigma-Aldrich, F3648), rabbit anti-p-Smad3 at 1:400 (Abcam, ab52903), mouse anti-embCMHC at 1:50 (N2.261; developed by H.M. Blau, obtained from Developmental Studies Hybridoma Bank), rabbit anti-Col1a1 at 1:200 (GeneTex, GTX124368) and guinea pig anti-ColXIIa1 at 1:1000 (Bader et al, 2009). Secondary antibodies (Jackson Immunoresearch) were used at 1:500 and Phalloidin 390 (ATTO, AD 390–81) was used at 1:500.
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