HeLa cells were grown on glass coverslips in 6-well plates and exposed to conditioned medium obtained from ADSC or serum-free medium during 24 h. Cells were then rinsed with 1X PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Subsequently, cells were blocked with PBS and 5% bovine serum albumin (Sigma-Aldrich) for 2 h and then cells were incubated overnight in a humid chamber with the corresponding primary antibody: RelB, p65, p52, Vimentin, Fibronectin, N-cadherin or E-cadherin (Thermo-Fisher). The cells were washed and incubated with a corresponding secondary antibody (#W4028 or #W4018 Promega) for 1 h. Finally, cells were washed with PBS and the slides were mounted in Everbrite mounting medium with DAPI (Biotium Inc., Hayward, CA, USA) and stored at 4 °C. The fluorescence analysis was performed in a confocal microscope (Zeiss LSM 510).
Everbrite mounting medium with dapi
Manufactured by Biotium
Sourced in United States
Everbrite mounting medium with DAPI is a specialized liquid mounting medium designed for use in fluorescence microscopy. It is formulated to preserve the fluorescent signal and prevent photobleaching of fluorescent dyes, including DAPI which stains cell nuclei. This product provides a clear, non-fading solution for mounting and preserving fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Fibronectin, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Thermo Fisher Scientific (1 mentions)
N cadherin, by Thermo Fisher Scientific (1 mentions)
Vimentin, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Citrisolv, by Thermo Fisher Scientific (1 mentions)
Casein, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Rabbit igg, by Vector Laboratories (1 mentions)
1 9 dimethylmethylene blue dmmb, by Merck Group (1 mentions)
Trueview, by Vector Laboratories (1 mentions)
Dnase, by Thermo Fisher Scientific (1 mentions)
Geneticin g418, by InvivoGen (1 mentions)
Cf594 donkey anti goat, by Biotium (1 mentions)
Proteinase k, by New England Biolabs (1 mentions)
Papain, by Merck Group (1 mentions)
L cysteine, by Merck Group (1 mentions)
Chondroitin sulfate, by Merck Group (1 mentions)
6 protocols using everbrite mounting medium with dapi
1
Immunofluorescence Analysis of Stem Cell Secretome Effects
2
Histological and Immunostaining Analysis of Spinal Tissues
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Sections were deparaffinized with SlideBrite (Thermo Fisher Scientific) and then dehydrated with ethanol prior to being stained using safranin-O with fast green and hematoxylin counterstains to examine the general morphology of the spine as well as the presence of glycosaminoglycan. To prepare samples for immunostaining, serial sagittal paraffin sections (10 µm) were deparaffinized in CitriSolv (Thermo Fisher Scientific) and rehydrated through a series of graded ethanols to distilled water. Next, samples were subjected to antigen retrieval (10 mM sodium citrate, pH 6.0) and blocking (1:500 dilution; 100 µL goat serum: 50 mL Tris-buffered saline, 0.1% Tween 20 (TBST) for 1 hour) steps were performed prior to going through TBST wash and antibody application steps. Antibodies conjugated to fluorophores for cell proliferation (Ki67—AF647, Clone # SolA15, eBioscience), a dural marker (α-SMA—AF647, Clone # 1A4, Biolegend), Hic1 (Clone # H6, Santa Cruz), Prx1 (Novus Biologicals) were applied at 4°C overnight. Sections were then washed 3 times at 10 minutes/wash in TBST and mounted using EverBrite Mounting Medium with DAPI (Biotium) for nuclear counterstaining and coverslipped.
3
Chondroitin Sulfate Immunofluorescence Protocol
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Dulbeccos Modified Eagles Medium and DNAse were purchased from Fisher Scientific, Pittsburgh, PA. Rabbit anti-chondroitin sulfate antibody was purchased from Bioss Antibodies Inc.,Woburn, MA. Normal goat IgG, rabbit IgG and TrueView were purchased from Vector Laboratories, Burlingame, CA. CF488 donkey anti-rabbit IgG, CF594 donkey anti-goat and EverBrite™ mounting medium with DAPI were purchased from Biotium, Fremont, CA. Biotin- and HRP-conjugated goat anti-human Apolipoprotein B antibodies were purchased from Academy Bio-Medical, Houston, TX. chondroitin sulfate monoclonal antibody (CS-56), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG were obtained from Life Technologies, Carlsbad, CA. Heparan Sulfate antibody (clone F58-10E4), was purchased from AMSBIO, Cambridge, MA and Alexa Fluor 488 together with Alexa Fluor 594 conjugated Donkey Anti-Mouse IgM from Jackson ImmunoResearch Laboratories, West Grove, PA. Papain, L-cysteine, chondroitin sulfate and 1,9-Dimethyl-Methylene Blue (DMMB) were all purchased from Sigma-Aldrich, Milwaukee, WI. ELISA-Bright HRP substrate, 24 and 96-well plates were obtained from VWR International, Batavia, IL. Casein and Poly-L-Lysine from EMD Millipore, Temecula, CA; G418 (Geneticin) from InvivoGen, San Diego, CA and Proteinase K from New England BioLabs, Ipswich, MA.
4
Visualization of DOX-loaded Platelets
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Loading of DOX in PLT was visualized by immunofluorescence staining and observed by deconvolution microscopy. Degreased coverslips were coated with 200 μL of 10 mg/ml fibrinogen (Sigma-Aldrich) in saline at 22 ± 2 °C for 30 min [38 (link)]. 2 × 108 DOX-loaded PLT were loaded onto the coverslip at 22 ± 2 °C for 30 min and fixed with 2 mL 4% paraformaldehyde for 10 min followed by blocking with 200 μL 2% bovine serum albumin (BSA; Sigma-Aldrich) in phosphate-buffered saline (PBS; Hyclone, Logan, UT, USA). CD41a PLT surface markers were stained with 200 μL of mouse anti-human CD41a-APC (BD Bioscience, San Jose, CA, USA) at a 1:20 dilution in 2% BSA for 1 h. The coverslips were mounted onto microscopic slides with 10 μL Everbrite mounting medium with DAPI (Biotium, Hayward, CA, USA) and visualized by DeltaVision Personal Deconvolution Microscopy (GE Healthcare, Marlborough, MA, USA). Three digital (3D) images were generated by Volocity 6.3 software (PerkinElmer, Waltham, MA, USA).
5
Immunoblotting and Immunofluorescence Analysis
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Rabbit anti-pEGFR (Y845), anti-pEGFR (Y1086), anti-pPYK2 (Y402), anti-pSTAT3 (Y705), anti-pSrc Family (Y416), anti-EGFR, anti-STAT3, anti-β-actin and Alexa 594-conjugated anti-rabbit F(ab)'2 fragment antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-Histone H3 antibodies were purchased from Thermo Scientific (Rockford, NY, USA). Isotype control IgG (MAB002) and anti-IL-17RB antibodies were purchased from R&D systems (Minneapolis, MN, USA). Iressa (Gefitinib) and the Src inhibitor AZM475271 were obtained from Tocris Bioscience (R&D systems). 7-AAD was purchased from Beckman (Coulter, France). EverBrite mounting medium with DAPI was purchased from Biotium (Hayward, CA, USA).
6
Immunofluorescent Staining of Clusterin and HMGB1
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DU145 tumor cells, grown in covered wells of a microscope slide, were treated with 55 nM DTX for 24 h at 37 °C. Then, the cells were fixed and dually stained with anti-clusterin and anti-HMGB1. Briefly, slides were blocked with goat serum, followed by incubation with mouse anti-human clusterin (Upstate, 1:500) and rabbit anti-human HMGB1 (Cell Signaling, 1:200) overnight at 4 °C. Next day, slides were incubated with an Alexa 549 anti-mouse secondary antibody (Invitrogen, 1:1000) or Alexa 488 anti-rabbit secondary antibody (Invitrogen, 1:1000) for 30 min. The slides were mounted with Everbrite mounting medium with DAPI (Biotium). Slides were analyzed with an automated Zeiss Imager Z.1 upright microscope through a 63×/1.4 NA objective with DAPI, FITC, and Alexa 549 filters. Images were captured by using the AxioCam MRm CCD camera and Axiovision (Version 4.7; Carl Zeiss).
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