The CGH experiments followed the methodology described in Symonová et al. (2015 ), with modifications. Briefly, prior to hybridization, slides were aged at 37°C for 2h, followed by an RNAse A (90 min, 37°C) and then pepsin (50 μg/ml in 10 mM HCl, 3 min, 37°C) treatments. Chromosomes were subsequently denatured in 75% formamide (pH 7.0) in 2 × SSC (74°C, 3 min), and then immediately cooled and dehydrated through 70% (cold), 85%, and 100% (RT) ethanol series. The hybridization mixture was denatured at 86°C for 6 min, cooled at 4°C (10 min) and then applied on the slides. The hybridization was performed at 37°C for 72 h. Post-hybridization washes were carried out once in 50% formamide in 2 × SSC (pH 7.0) (44°C, 10 min each) and three times in 1 × SSC (44°C, 7 min each). Prior to probe detection, the slides were incubated with 3% non-fat dried milk (NFDM) in order to avoid the non-specific binding of antibodies. The hybridization signal was detected using Anti-Digoxigenin-Rhodamin (Roche) and Avidin-FITC (Sigma). Chromosomes were counterstained and mounted in antifade containing 1.5 μg/ml DAPI (Vector, Burlingame, CA, USA).
Avidin fitc
Manufactured by Merck Group
Sourced in United States
Avidin-FITC is a laboratory reagent that consists of the protein avidin conjugated to the fluorescent dye FITC (fluorescein isothiocyanate). Avidin has a high affinity for the vitamin biotin, and the FITC label allows for the detection and visualization of biotin-labeled molecules.
Automatically generated - may contain errors
Lab products found in correlation
Anti digoxigenin rhodamin, by Roche (6 mentions)
Antifade solution, by Vector Laboratories (4 mentions)
Anti digoxigenin rhodamine, by Roche (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Ab104224, by Abcam (2 mentions)
Ab41489, by Abcam (2 mentions)
Ab39374, by Abcam (2 mentions)
Ab36001, by Merck Group (2 mentions)
Glutathione sepharose 4b resin, by Cytiva (2 mentions)
Sc 188, by Santa Cruz Biotechnology (2 mentions)
Pgex 4t vector, by Cytiva (2 mentions)
Af416, by Santa Cruz Biotechnology (2 mentions)
Donkey anti mouse igg conjugated alexa fluor 488 or 594, by Thermo Fisher Scientific (2 mentions)
Goat anti chicken igg conjugated alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Donkey anti rabbit igg conjugated alexa fluor 488 or 594, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 conjugated donkey anti goat, by Thermo Fisher Scientific (2 mentions)
Fluorescence microscope, by Olympus (1 mentions)
N134 12, by R&D Systems (1 mentions)
Biotin conjugated ib4, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
19 protocols using avidin fitc
1
Comparative Genomic Hybridization Protocol
2
Histological Analysis of Aortic Inflammation
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Serial cryosections of the ascending aorta were mounted on glass slides, air-dried, blocked with 10% goat serum in PBS and incubated overnight at 4°C with biotinylated anti-VCAM-1 (1:50 dilution in PBS, eBiosciences), biotinylated anti-ICAM-1 (1:50 dilution in PBS, LifeSpan Biosciences) or FITC-labeled anti-P-selectin (1:50 dilution in PBS, Santa Cruz) antibodies. Avidin-FITC (1:500 dilution in PBS, Sigma) was added to bind to the biotinylated antibodies and incubated for 1 h at 37°C. Sections were imaged using a fluorescence microscope (Olympus, Tokyo, Japan).
3
CGH Protocol with Modifications
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For the CGH experiments, we used the methodology described in77 , with several modifications. Briefly, a thermal aging of slides was performed prior to hybridization, at 37 °C for 2 h. Next, a treatment with RNase A (100 µg/ml, 90 min, 37 °C) took place, followed by the pepsin digestion (50 µg/ml in 10 mM HCl, 3 min, 37 °C). Denaturation of chromosomes was done in 75% formamide/2 × SSC at 74 °C for 3 min, and slides were then immediately dehydrated in 70% (cold), 85%, and 100% (RT) ethanol. The probe cocktail was denatured at 86 °C for 6 min, chilled on ice for 10 min and then applied to each slide. The hybridization was performed at 37 °C for 3 days in a dark humid chamber. Subsequently, non-specific hybridization was removed by stringent washing: once or twice in 50% formamide/2 × SSC (44 °C, 10 min each) and three times in 1 × SSC (44 °C, 7 min each). To block non-specific binding sites for antibodies, slides were incubated with 3% non-fat dried milk (NFDM) at 37 °C and subsequently hybridization signals were detected using Anti-Digoxigenin-Rhodamin (Roche) and Avidin-FITC (Sigma). Finally, the preparations were mounted in antifade containing 1.5 µg/ml DAPI (Vector).
4
Generating Kcns1 Antibody via Fusion Protein
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To generate Kcns1 antibody, the C-terminal end region of mouse Kcns1 (aa 469-497, NP_032461.2) was PCR amplified and subcloned into pGEX-4T vector (glutathione S-transferase fusion protein, Amersham Pharmacia Biotech). Glutathione S-transferase fusion proteins were purified using glutathione-Sepharose 4B resin (Amersham Pharmacia Biotech) and used to immunize rabbit. Kcns1 antibody was affinity purified using glutathione S-transferase fusion protein. Primary antibodies and their dilutions used in this study were: rabbit anti-Kcns1 (1:2000), mouse anti-NeuN (1:1000, Abcam #ab104224), chicken anti-β3tubulin (1:1000, Abcam #ab41489), chicken anti-peripherin (1:500, Abcam #ab39374), goat anti-CGRP (1:1000, Abcam #ab36001), chicken anti-NF200 (1:1000, Merck Millipore AB5539), mouse anti-Nav1.8 (1:1000, Neuromab N134/12), goat anti-CSF1 (1:1000, R&D Systems #AF416), and rabbit anti-ATF3 (1:500, Santa Cruz Biotechnology #sc-188). Secondary antibodies used were donkey anti-mouse IgG-conjugated Alexa Fluor 488 or 594, goat anti-chicken IgG-conjugated Alexa Fluor 594, donkey anti-rabbit IgG-conjugated Alexa Fluor 488 or 594, and donkey anti-goat–conjugated Alexa Fluor 594 (all 1:1000, Invitrogen). IB4 binding was visualized using biotin-conjugated IB4 (1:200, Sigma L2140) and Avidin-FITC (1:500, Sigma).
5
Fluorescence In Situ Hybridization Protocol
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Fluorescence in situ hybridization (FISH) experiments were performed as described in Yano et al. (2017) [25 ], with slight modifications. The experiment was performed under high stringency conditions on mitotic chromosome spreads. Metaphase chromosome slides were incubated with RNase (40 μg/ml) for 1.5 h at 37 °C. After denaturation of the chromosomal DNA in 70% formamide/2× SSC at 72 °C for 3 min, the hybridization mixture (2.5 ng/μl probes, 2 μg/μl salmon sperm DNA, 50% deionized formamide, 10% dextran sulphate) was dropped on the slides, and the hybridization was performed overnight at 37 °C in a moist chamber containing 2× SSC. The first post-hybridization wash was performed with 2× SSC for 5 min at 42 °C, and a final wash was performed at room temperature in 1× SSC for 5 min. The signal detection was performed using anti-digoxigenin rhodamine (Roche) for the 18S rDNA probe and with avidin-FITC (Sigma) for 5S rDNA. Subsequently, the slides were dehydrated again in an ethanol series (70%, 85% and 100%), 2 min each. Finally, the slides were counterstained with DAPI and mounted in an antifading solution (Vectashield from Vector Laboratories).
6
Investigating Cellular Signaling Pathways
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The MEK inhibitor PD184352, the AKT inhibitor AKTi, Epidermal Growth Factor (EGF), basic Fibroblast Growth Factor (bFGF), Polyethylenimine (PEI), Avidin-FITC and 4′6-diamino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (Rehovot, Israel). Protein A/G PLUS-agarose beads were obtained from Santa Cruz Biotechnology, Inc. (CA, USA). Glutathione beads were purchased from GE-healthcare and Proximity ligation assay kit was from Olink Bioscience (both Uppsala, Sweden). Albumin bovine serum (BSA) was purchased from MP biomedical (OH, USA). Si RNAs and Dharmafect were from Thermo Fisher Scientific (CO, USA). NBT/BCIP developing substrates were purchased from Promega (Wis, USA). Soybean trypsin inhibitor (SBTI) was purchased from Biological Industries (Beit HaEmek, Israel).
7
Biomaterial Functionalization via PEG-dAAm
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PEG-dAAm
3.7 kDa, 1-ethyl-3-(3-dimethyl-aminopropyl)
carbodiimide hydrochloride (EDC), 2-mercaptoethanol, 2-(N-morpholino)ethanesulfonic acid (MES), phenylalanine ammonia lyase
(PAL), glucose oxidase (GOx), avidin-FITC, IgG-FITC, and bovine serum
albumin (BSA)-FITC were purchased from Sigma-Aldrich. Sodium carbonate
(NaCO3), calcium chloride (CaCl2), N-hydroxysuccinimide (NHS), sodium bicarbonate (NaHCO3),
and hydrochloric acid (HCl) were purchased from Fisher Scientific.
Irgacure 2959 was purchased from BASF. Acrylate-PEG-NH-Boc MW 2 kDa
and PEG-dAAm (0.6, 2, 10, and 20 kDa) were purchased from Creative
PEGWorks. Cy5-NHS was synthesized as previously reported.18 (link)
3.7 kDa, 1-ethyl-3-(3-dimethyl-aminopropyl)
carbodiimide hydrochloride (EDC), 2-mercaptoethanol, 2-(N-morpholino)ethanesulfonic acid (MES), phenylalanine ammonia lyase
(PAL), glucose oxidase (GOx), avidin-FITC, IgG-FITC, and bovine serum
albumin (BSA)-FITC were purchased from Sigma-Aldrich. Sodium carbonate
(NaCO3), calcium chloride (CaCl2), N-hydroxysuccinimide (NHS), sodium bicarbonate (NaHCO3),
and hydrochloric acid (HCl) were purchased from Fisher Scientific.
Irgacure 2959 was purchased from BASF. Acrylate-PEG-NH-Boc MW 2 kDa
and PEG-dAAm (0.6, 2, 10, and 20 kDa) were purchased from Creative
PEGWorks. Cy5-NHS was synthesized as previously reported.18 (link)
8
Chromosome analysis by CGH
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CGH experiments were performed according to Symonová et al. [53 ]. The slides were incubated at 37 °C in a dark humid chamber for 72 h. The hybridization signal was detected with anti-digoxigenin-Rhodamin (Roche) diluted in 0.5% bovine serum albumin (BSA) in phosphate-buffered saline solution (PBS), and avidin-FITC (Sigma, St Louis, MO, USA) diluted in PBS containing 10% normal goat serum (NGS). The chromosomes were counterstained with DAPI (1.2 µg/mL) and mounted in an antifade solution (Vector, Burlingame, CA, USA).
9
Immunofluorescence Microscopy of Tubulin
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Cells were fixed with 4% paraformaldehyde dissolved in D‐PBS for 30 min at 4 °C. After washing with D‐PBS, cells were treated with 0.1% Triton X‐100 in D‐PBS for 3 min and in turn incubated with 1% bovine serum albumin in D‐PBS for 15 min. Cells were reacted with avidin conjugated with fluorescein isothiocyanate (avidin‐FITC, Sigma‐Aldrich) and anti‐α‐tubulin rabbit polyclonal antibody (Abcam, Cambridge, UK) overnight at 4 °C. After washing with D‐PBS, cells were incubated with an anti‐rabbit antibody conjugated with Alexa 594 and mounted in Vectashield mounting medium (Vecror Laboratories, Burlingame, CA, USA) containing 4′,6‐diamidino‐2‐phenylindole (DAPI). Fluorescence was observed using an FV1200 laser scanning confocal microscope (Olympus, Tokyo, Japan) and analyzed with fluoview software (Olympus).
10
Dual-Color FISH Analysis of Chromosomes
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Fluorescence in situ hybridization (FISH) was performed on mitotic chromosome spreads [51 (link)] and dual-color FISH analysis was carried out using 5S rDNA and 18S rDNA probes. Briefly, the metaphase chromosome slides were incubated with RNAse (40 µg/mL) for 1.5 h at 37°C. After denaturation of chromosomal DNA in 70% formamide (pH 7.0), the spreads were incubated in 2xSSC for 4 min at 70°C. Hybridization mixtures containing 100 ng of the denatured probe, 10 mg/mL dextran sulfate, 2xSSC, and 50% formamide (pH 7.0) in a final volume of 30 µL were applied to the slides. Hybridization was conducted overnight at 37°C in a 2xSSC moist chamber. Posthybridization washes were carried out at 37°C in 2xSSC, 50% formamide (pH 7.0) for 15 min, followed by a second wash in 2xSSC for 15 min, and a final wash at room temperature in 4xSSC for 15 min. Signal detection was performed using avidin-FITC (Sigma, St. Louis, MO, USA) for the 5S rDNAprobe and anti-digoxigenin-rhodamine (Roche, Mannheim, Germany) for the 18S rDNA probe. Posthybridization washes were carried out in a shaker (150 rpm) and chromosomes were counterstained with DAPI (1.2 µg/mL).
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