Human ventricular samples were fixed in 4% paraformaldehyde (Santa Cruz) in PBS (Lonza) and processed for paraffin embedding. Paraffin-embedded sections (6 μm thick) were de-waxed in xylene and rehydrated in ascending alcohols. The immunofluorescence analysis was performed following antigen retrieval with incubation with target retrieval solution citrate pH 6/microwave (Dako). Sections were incubated at 4°C overnight with primary antibodies for the detection of mesenchymal surface markers (see Supplementary Table S2 ), namely, anti-CD29 (1:40; Leica), anti-CD44 (1:200; Abcam), and anti-CD105 (1:100; Abcam) diluted in 2% goat serum (Sigma–Aldrich). After washing with PBS, sections were incubated for 1 h at RT in the dark with proper secondary antibodies (see Supplementary Table S3 ). Nuclear staining was performed by incubating sections with Hoechst 33342 (1:1000; Life Technologies). Sections were observed by Zeiss Axio Observer.Z1, with Apotome technology, and images acquired with the software AxioVision Rel. 4.8. For each explanted heart patient, five slices and at least 10 fields for each slice were examined.
Anti cd44
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-CD44 is a monoclonal antibody that recognizes the CD44 cell surface glycoprotein. CD44 is a receptor for hyaluronic acid and functions in cell-cell interactions, cell adhesion, and migration.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd133, by Abcam (10 mentions)
Anti β actin, by Abcam (5 mentions)
Anti e cadherin, by Cell Signaling Technology (5 mentions)
Anti oct4, by Abcam (5 mentions)
Anti cd90, by Abcam (5 mentions)
Anti cd24, by Abcam (5 mentions)
Anti vimentin, by Cell Signaling Technology (5 mentions)
Anti epcam, by Abcam (5 mentions)
Anti β catenin, by Abcam (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Anti cd29, by Abcam (4 mentions)
Anti β actin, by Santa Cruz Biotechnology (4 mentions)
Anti cd105, by Abcam (3 mentions)
Anti n cadherin, by Cell Signaling Technology (3 mentions)
Anti sox2, by Abcam (3 mentions)
Anti aldh1, by Cell Signaling Technology (3 mentions)
Anti cd166, by Abcam (3 mentions)
Fluorchem fc2, by Bio-Techne (3 mentions)
Anti mouse secondary antibodies, by Abcam (3 mentions)
Anti β actin antibody, by Abcam (3 mentions)
65 protocols using anti cd44
1
Immunofluorescence Analysis of Mesenchymal Markers
2
Western Blot Analysis of Stem Cell Markers
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The tumor cells were lysed by using RIPA buffer with Phenyl methane sulfonyl fluoride (PMSF). The same amount of protein was separated with 10% SDS-PAGE gel and then was transferred onto PVDF membranes (Biotech). Subsequently, 5% skim milk (diluted in PBS) was used to blocked the PVDF membranes for 2 h at RT. And then the membranes were incubated with anti-Gli1 (Santa), anti-CD44 (Abcam), anti-LSD1 (ZSGB-BIO), anti-Sox9 (Abcam), anti-β-actin (Abcam). The next step is to incubate anti-rabbit/mouse for 2 h. Detection was performed by the ECL kit.
3
Western Blot Analysis of Protein Targets
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Western blot assay was carried out as previously described.24 Cells were washed with PBS and then lysed with RIPA lysis buffer (Solarbio, China) and protease inhibitors (Roche Applied Science, Indianapolis, Switzerland). The protein concentration was measured using the bicinchonininc acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal amounts of protein were subjected to 10% SDS‐polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were subsequently blocked with 5% non‐fat milk for 2 hours and incubated with primary antibodies overnight at 4°C. The primary antibodies used were anti‐MGMT (1:500, Abcam, Cambridge, UK) and anti‐Survivin (1:1000, Abcam), anti‐β‐catenin, anti‐CD44, anti‐C‐Jun, anti‐C‐Myc, anti‐cyclinD1, anti‐LEF1, anti‐TCF1/TCF7, anti‐MMP7, anti‐Axin2, anti‐Met, anti‐PARP, anti‐caspase‐3, anti‐BAX, anti‐Bcl2, anti‐cleaved‐caspase‐3, anti‐E‐cadherin, anti‐N‐cadherin and anti‐Vimentin (these primary antibodies are all: 1:1000, Cell Signaling Technology, Boston, MA, USA), as well as anti‐GAPDH, and secondary antibodies were HRP‐conjugated goat anti‐mouse or goat anti‐rabbit IgG antibody (1:1000, Beyotime). All experiments were performed in triplicate.
4
Western Blot Analysis of Stem Cell Markers
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Harvested cells were lysed by RIPA buffer. After sonication, samples were centrifuged at 12 000 g for 15 min at 4°C. Total protein concentration was determined by applying DC Protein Assay Kit I (Bio‐Rad, USA). Proteins were transferred to Hybond nitrocellulose membranes (USA) after separation on 12% SDS‐PAGE. 5% skim milk was added to seal the membrane in Tris‐buffered saline (pH 7.5) at room temperature. The membrane was incubated with the primary antibody overnight at 4°C, followed by 4‐h incubation with the secondary antibody at room temperature. Protein bands were developed by ECL kit (Millipore, USA). Protein levels were assessed by ImageJ (USA). Primary antibodies including anti‐CD133, anti‐CD44, anti‐Oct‐4, anti‐HK2, anti‐PKM2, anti‐LDHA, anti‐β‐actin, and secondary antibody anti‐IgG were purchased from Abcam (UK).16
5
Western Blot Characterization of Cells
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Total protein from brain tissue or BV2 cells was collected, and protein concentrations were determined with a BCA kit (Beyotime Institute of Biotechnology, China) following the manufacturer’s guidelines. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Millipore, MA, USA), blocked with 5% skim milk, and incubated with primary antibodies (see below) either overnight at 4 °C or for 1 h at room temperature. The membranes were washed and incubated with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:500; Beyotime Institute of Biotechnology, China) and were visualized using an ECL Plus kit (Millipore). Densitometry was performed to quantify the signal intensity using ImageJ software (Version 1.45 J; National Institutes of Health, Bethesda, MD, USA). The primary antibodies were rabbit anti-Nrf2, anti-HO-1, anti-NF-κB1, anti-CD29, anti-CD90, anti-CD44, anti-CD105, anti-CD34, anti-vWF, anti-BDNF, anti-TrkB, and anti-GAPDH (Abcam, Cambridge, UK).
6
Immunofluorescence Analysis of CD44 and CD24 in MDA-MB-231 Cells
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Briefly, MDA-MB-231 cells were seeded on coverslips in 35 mm dishes and treated with siRNA respectively for 24 h, then cells were fixed with 4% paraformaldehyde. Fixed cells were incubated with anti-CD44 (1:100, Cat No. 6124) and CD24 antibodies (1:100, Cat No. 64064) (Abcam, Hangzhou, China) and then the fluorochrome-tagged secondary antibody (1:500, FITC-conjugated anti-rabbit, TRITC-conjugated anti-mouse) (Abcam, Hangzhou, China). Following stained with Hoechst33342 in PBS buffer, coverslips were mounted on slides.
7
Comprehensive Antibody Panel for Cellular Analyses
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The following antibodies were purchased: for Western blotting, anti-β-catenin (1:5000; Abcam, cat. no. ab32572), anti-β-actin (1:5000; Bioss, cat. no. bs-0061R), anti-CDK5RAP2 (1:2000; Abcam, cat. no. ab70213), anti-survivin (1:2000; Abclonol, cat. no. A1551), anti-ALDH1 (1:1000; Cell Signaling Technology, cat. no. 54135), anti-Notch1 (1:1000, Abcam, cat. no. ab52627), anti-EZH2 (1:1000; Cell Signaling Technology, cat. no. 5246), anti-CCND1 (1:1000, Cell Signaling Technology, cat. no. 55506), and horseradish peroxidase-conjugated secondary antibodies (1:1000; Cell Signaling Technology, Inc.); for IHC analyses, anti-CDK5RAP2 (1:400; Abcam, cat. no. ab235893), anti-ALDH1 (1:50; Abcam, cat. no. ab52492), anti-SOX2 (1:100; Abcam, cat. no. Ab92494), anti-CD44 (1:4000; Abcam, cat. no. ab189524), anti-CD133 (1:1000; Abcam, cat. no. ab222782), anti-Notch1 (1:150; Abcam, cat. no. ab52627), anti-EZH2 (1:200; Cell Signaling Technology, cat. no. 5246), and anti-CCND1 (1:200; ABclonal, cat. no. A19038); and for immunofluorescence analyses, anti-α-tubulin (1:500, YL1/2; Santa Cruz Biotechnology, cat. no. sc-53029), anti-γ-tubulin (1:1000, GTU88; Sigma-Aldrich, cat. no. T5326), and Alexa Fluor-conjugated secondary antibodies (1:500; Thermo Fisher Scientific).
8
Immunohistochemical Analysis of Tumor Markers
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Paraffin-embedded tumor and organ samples were sectioned, placed on slides, dewaxed, rehydrated, pretreated with hydrogen peroxide, washed with phosphate-buffered saline, and then stained with H&E, followed by rinsing and mounting under coverslips using Permount (Fisher Scientific, San Francisco, CA, USA). For IHC staining, endogenous peroxidase was blocked with 3% hydrogen peroxide, and the tissue samples on slides were then incubated with primary anti-human antibodies, including anti-hypoxia-inducible factor (HIF)-1α (catalog no. ab5168; Abcam, Cambridge, UK), anti-CD24 (catalog no. ab31622; Abcam), and anti-CD44 (catalog no. ab189524; Abcam). Color was visualized using an SPlink Detection Kit (catalog no. SP-9000; ZSGB-BIO, Guangzhou, China) in accordance with the manufacturer’s instructions. TUNEL assay was performed using an in situ cell death detection kit (catalog no. 11684817910; Roche Applied Science, Mannheim, Germany) according to the manufacturer’s protocol. All slides were analyzed and photographed by an experienced pathologist.
9
Immune Phenotyping of hMSCs with CYTL1 Manipulation
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To identify the immune phenotypes of hMSCs subjected to CYTL1 overexpression or knockdown, FACS was performed. hMSCs were infected with 400 MOI of Ad-C or Ad-CYTL1 for 2 h and cultured for an additional 48 h. Alternatively, cells were infected with 25 MOI of control lentivirus shRNA or the two different CYTL1 shRNAs. shRNA-infected hMSCs were selected by puromycin. hMSCs infected with Ad-CYTL1 or the lentivirus-based CYTL1 shRNA were suspended in PBS and incubated for 30 min with anti-CD44, anti-CD29, or anti-CD90 (Abcam). The cells were washed and incubated with goat anti-mouse Alexa Fluor 488 or goat anti-rabbit Alexa Fluor 594 (Invitrogen). The cells were washed and immediately examined using a FACS Canto II flow cytometer (BD Biosciences). The data were analyzed with the FlowJo software (Tree Star).
10
Immunoblot Analysis of Cell Lysate Proteins
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Cell lysate preparation and immunoblot analyses were performed as previously reported [40 (link)]. Filters were probed with the indicated primary antibodies: anti-TIMP-1 and anti-CD99 (R&D Systems, Minneapolis, MN, USA); anti-integrin β1, anti-CD63 and anti-vinculin (Santa Cruz Biotechnology, Dallas, TX, USA); anti-vimentin, anti-integrin αv, anti-PDGFRβ, anti-pAKT, anti-pErk 1/2 and anti-ZO-1 (Cell Signaling Technology Inc., Danvers, MA, USA); anti-ITPRIPL1 (OriGene Technologies, Rockville, MD, USA); anti-NF-kB-p65 (Elabscience, Houston, TX, USA); anti-actin (Sigma-Aldrich); and anti-CD44 (Abcam, Cambridge, UK). Densitometric analysis was performed on at least two different exposures to assure the linearity of each acquisition using ImageJ software (v1.46r).
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