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.
Anti cd24
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-CD24 is a lab equipment product used for the detection and analysis of CD24 (Cluster of Differentiation 24), a cell surface glycoprotein. It can be used in various research applications that require the identification and study of CD24-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd44, by Abcam (5 mentions)
In situ cell death detection kit, by Roche (2 mentions)
Anti collagen 2, by Abcam (2 mentions)
Anti aggrecan, by Abcam (2 mentions)
Splink detection kit, by ZSGB-BIO (2 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Hif 1α, by Abcam (1 mentions)
Anti arginase 1, by Abcam (1 mentions)
Anti cd45, by Abcam (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Anti cd68, by Abcam (1 mentions)
Anti inos, by Abcam (1 mentions)
Anti cd11b, by Abcam (1 mentions)
Confocal fluorescence microscope, by Leica camera (1 mentions)
Anti phos smad2, by Cell Signaling Technology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Anti epcam, by Abcam (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti rabbit immunoglobulin g igg anti mouse igg, by Merck Group (1 mentions)
11 protocols using anti cd24
1
Immunohistochemical Analysis of Tumor Markers
2
Investigating p38 MAPK Signaling Pathways
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The anti-p38α and p-p38 used in western blot (WB) or immunoprecipitation (IP) or immunofluorescence (IF), and anti-p38β, anti-p38γ, anti-p38δ used in WB or IP, and anti-β-actin, anti-MKK-3, anti-MKK-4, anti-MKK-6 used in WB were purchased from CST. The anti-p38βand CD206 were purchased from ST, and anti-p38γ and anti-p38δ were from RD, the anti-CD68, anti-CD45, anti-CD24, anti-CD11b, anti-iNOS and anti-Arginase-1 were obtained from Abcam were used in IF. Anti-IFNγ and anti-GM-CSF uesd in neutralising assay were purchased from Abcam. BDTM Flow Cytometric Bead (CBA) Array flex set (Human IFNγ GM-CSF, TNF-α, IL-1β, MCP-1 flex set) was purchased from BD Biosciences. Plasmids short hairpin RNA for p38α, p38β p38γ and p38δ in a lentiviral GV118 vector were obtained from Genechem (Genechem Shanghai, China). IL-1β was purchased from PeproTech.
3
Immunocytochemical Analysis of Nodal Signaling in Colorectal Cancer Cells
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For immunocytochemistry, SW480 cells, LOVO cells, HCT116 cells, CD24-positive and -negative HCT116 cells, and human colon cancer stem cells cultured without or with Nodal were fixed with 4% paraformaldehyde (PFA) and permeabilized in 0.4% triton-X 100 (Sigma-Aldrich) for 45 min. After washing twice with phosphate-buffered saline (PBS), cells were blocked in 1% BSA for 30 min and followed by incubation with primary antibodies at a dilution with 1 : 100 overnight at 4°C. Primary antibodies included anti-NODAL (Abcam), anti-ALK-4 (Santa Cruz Biotechnology), anti-ALK-7 (Santa Cruz), anti-ACTR-IIB (Santa Cruz), anti-CD24 (Abcam), anti-CD44 (Abcam), anti-phos-Smad2, and anti-phos-Smad3 (Cell Signaling Inc). After three washes with PBS, the cells were incubated with the secondary antibody, including FITC-conjugated or rhodamine-conjugated IgG (Jackson ImmunoResearch Laboratories) at a 1 : 200 dilution for 1 hour at room temperature. DAPI (4′-6-diamidino-2-phenylindole) was used to stain cellular nuclei, and the cells were observed for epifluorescence using a confocal fluorescence microscope (Leica). Immunocytochemistry was performed in triplicate and representative images were presented.
4
Western Blot Analysis of Cell Signaling Proteins
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Cells and tissues were harvested in sampling buffer [62.5 mmol/L Tris-HCl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate (SDS)] and heated for 5 minutes at 100°C. Protein concentration was determined with the Bradford assay using a commercial kit purchased from Bio-Rad Laboratories (Hercules, CA, USA). Equal quantities of protein were separated electrophoretically on 10% SDS/polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Roche, Basel, Switzerland). The membranes were probed with diluted antibody. The expression of target proteins was determined with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG)/anti-mouse IgG (Sigma-Aldrich, St Louis, MO, USA) and enhanced chemiluminescence (Pierce, Rockford, IL, USA) according to the manufacturers' suggested protocols. The membranes were stripped and reprobed with an anti-β-actin mouse monoclonal antibody (Sigma-Aldrich) as a loading control. The related antibodies were anti-phosphorylated(p)-Smad3, anti-E-cadhernin, anti-Vimentin, anti-β-catenin (Cell Signaling Technology, Beverly, MA, USA), anti-CD24, anti-CD44, anti-CD166, anti-EpCam, anti-EF-1, anti-p84 anti-FRAT2, anti-LRP6, anti-FZD4, anti-FZD5 (Abcam, Cambridge, MA, USA) and anti-CD133 (Epitomics, USA).
5
In Situ Hybridization and Immunofluorescence
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An in situ hybridization kit was used (Cat No. GF002, Servicebio, China) according to the manufacturer’s instructions. In brief, following deparaffinization and rehydration, sections were subjected to digestion with proteinase K for 15 min at 37 °C. Then, the sections were hybridized to the FISH probe (500 nM) at 40 °C overnight, and finally, the signal probe hybridization solution was added for 45 min at 40 °C. After washing with SSC buffer, the sections were blocked with 5% BSA at room temperature for 30 min. The subsequent IF protocols were performed as previously described. The primary antibodies included anti-CD24 (1:100; Cat No. ab202073, Abcam, UK) and anti-HIF-1α (1:200; Cat No. 79233, CST, USA), and the corresponding secondary antibodies were Cy5-conjugated goat anti-rabbit IgG (H + L) (1:400; Cat No. GB27303, Servicebio, China) and Cy5-conjugated goat anti-mouse IgG (H + L) (1:400; Cat No. GB27301, Servicebio, China), respectively. The FISH probe for IL21-AS1 was designed and synthesized by Servicebio, and the probe sequences were 5′-CCATTCCTCTTCAGACTTCTACCCCT-3′ and 5′-AGTTTTAGACCTGGCACAGTTTTCCC-3′.
6
CD24 Expression in OC Cell Lines
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OC cell lines were cultured in confocal dishes (Cat No. BS-20-GJM, Biosharp, China) and then fixed with 4% paraformaldehyde in PBS for 15 min. The cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min and then incubated in 5% BSA for 1 hr. The cells were then incubated overnight at 4 °C with anti-CD24 (1:100; Cat No. ab202073, Abcam, UK). The secondary antibody anti-rabbit IgG (H + L) Alexa Fluor 555 (Cat No. ab150074, Abcam, UK) was used at a 1:500 dilution for 1 h. Antifade mounting medium with DAPI (Cat No. C1002, Beyotime, China) was used for nuclear staining. Then, the cells were imaged on a Leica confocal microscope.
7
Investigating EMT and Signaling Pathways
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DSF was purchased from Sigma (USA) and dissolved in DMSO. TGF-β was purchased from Peprotech (USA) and dissolved in 10 mM citric acid, PH3.0 to a concentration of 0.1–1.0 mg/ml. U0126 was purchased from Selleck Chemicals (USA) and dissolved in 10% DMSO, 30% Cremophor EL and 60% PBS to a concentration of 10 mM.
The following primary antibodies were used: anti-E-cadherin (WB: 1:1000, IF:1:200, IHC:1:400 dilution, CST), anti-vimentin (WB: 1:1000, IF:1:100, IHC:1:100 dilution, CST), anti-N-cadherin (WB: 1:1000 dilution, CST), anti-Snail (WB: 5 ng/ml, IF:10 μg/ml, IHC: 10 μg/ml, R&D Systems), anti-NF-κB/p65 (WB: 1:1000, IF:1:50, IHC:1:800 dilution, CST), anti-ERK (WB: 1:500, IF:1:50, IHC:1:50, dilution, Boster, Wuhan, China), anti-p-ERK (WB: 1:1000, IF:1:200, IHC:1:400 dilution, CST), anti-IκB-α (WB:1:500, IF: 1:50, IHC:1:50 dilution, Anbo, USA), anti-CD24 (WB: 1:200, IF:1:50 dilution, Santa Cruz), anti-CD24 (IHC:1:50 dilution, Abcam), anti-CD44 (WB:1:1000, IF:1:200, IHC:1:50 dilution, CST), anti-MMP-1 (WB:1:200 dilution, Santa Cruz), anti-MMP-3 (WB:1:200 dilution, Santa Cruz).
The following primary antibodies were used: anti-E-cadherin (WB: 1:1000, IF:1:200, IHC:1:400 dilution, CST), anti-vimentin (WB: 1:1000, IF:1:100, IHC:1:100 dilution, CST), anti-N-cadherin (WB: 1:1000 dilution, CST), anti-Snail (WB: 5 ng/ml, IF:10 μg/ml, IHC: 10 μg/ml, R&D Systems), anti-NF-κB/p65 (WB: 1:1000, IF:1:50, IHC:1:800 dilution, CST), anti-ERK (WB: 1:500, IF:1:50, IHC:1:50, dilution, Boster, Wuhan, China), anti-p-ERK (WB: 1:1000, IF:1:200, IHC:1:400 dilution, CST), anti-IκB-α (WB:1:500, IF: 1:50, IHC:1:50 dilution, Anbo, USA), anti-CD24 (WB: 1:200, IF:1:50 dilution, Santa Cruz), anti-CD24 (IHC:1:50 dilution, Abcam), anti-CD44 (WB:1:1000, IF:1:200, IHC:1:50 dilution, CST), anti-MMP-1 (WB:1:200 dilution, Santa Cruz), anti-MMP-3 (WB:1:200 dilution, Santa Cruz).
8
Immunohistochemical Characterization of iPSC Derivatives
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Prior to immunohistochemical staining, undifferentiated iPSCs and their differentiated derivatives, as well as NP positive control cells, were firstly fixed in cold acetone for 30 min at 4°C. Cells were then rinsed with PBS three times and blocked in 10% normal goat serum for 20 min. Samples were incubated with primary antibodies (Anti-Collagen II, Anti-Aggrecan, Anti-CD24, Abcam) overnight at 4°C, then rinsed with PBS three times, incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Invitrogen) for 2h at room temperature, rinsed again with PBS three times and then developed by use of the DAB kit. For the analysis of glycosaminoglycan, cells were fixed with formaldehyde, and stained with Toluidine blue. All immages were captured using an IX-71 fluorescence microscope (Olympus,Japan).
9
Protein Expression Analysis of Differentiated Cells
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After differentiation, the cells were collected and lysed in Tris–HCl buffer 0.05 mol/L (pH 8.0) containing 0.15 mol/L sodium chloride, 0.02% sodium azide, 0.1% SDS, 1% nonidet P (NP-40), 11 μg/mL aprotinin, and 0.1 mol/L phenylmethyl sulfonylfluoride (PMSF) (all reagents from Sigma Aldrich, USA). NP cells were used as positive control. The cell lysates were centrifuged at 12,000×g for 5 min at 4°C. SDS-PAGE was performed using 100μg total protein aliquots together with prestained molecular weight standards. Proteins were transferred onto nitrocellulose membranes, and then blocked with dried skimmed milk powder in Tween PBS-buffered saline (PBS-T) for 1 h at room temperature. Membranes were probed with primaryantibodies (Anti-CollagenI I, Anti-Aggrecan, Anti-CD24, 1:1000, Abcam) in PBS-T overnight at 4°Cand then horseradish peroxidase-conjugated secondary antibodies were incubated with the protein blots for 1 h at room temperature. Immunoreactive protein was detected using ECL chemiluminescence and images captured by the LAS-3000 imaging system (Fujifilm).
10
Signaling Pathways Regulating Stemness
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Primary antibodies used in this study were as follows: anti-Actin (Abcam, ab179467), anti-CDK1 (Abcam, ab133327), anti-STAT3 (Abcam, ab109085; Abcam, ab32500), anti-CD44 (Abcam, ab50137), anti-CD24 (Abcam, ab179821), anti-Sox2 (Cell Signailing Technology, #23064; Abcam, ab92494), anti-Oct4 (Abcam, ab181557; Cell Signailing Technology, #2890), anti-Nanog (Cell Signailing Technology, #4903), anti-HDAC3 (Abcam, ab32369), anti-Myc-Tag (Cell Signailing Technology, #2276), anti-Phospho-CDK1 (Tyr15) (Abcam, ab47594; Cell Signailing Technology, #4539), anti-Phospho-CDK1 (Thr14) (Cell Signailing Technology, #2543); anti-Phospho-CDK1 (Thr161) (Cell Signailing Technology, #9114); anti-Phosoho-STAT3 (Tyr705) (Abcam, ab76315; Affinity, AF3293), anti-Phospho-STAT3 (Ser727) (Abcam, ab32143), anti-Acetyl-lysine (PTM Bio, PTM-105; PTM Bio, PTM-101; Cell Signaling Technology, #9441S), anti-Ubiquitin (Abcam, ab134953); FITC-CD44 (BD Biosciences, catlog.555478), PE-CD24 (BD Biosciences, catlog.555428), Fisetin was purchased from Selleck (catalog.S2298). Trichostatin A (TSA) and Nicotinamide (NAM) was purchased from MCE (catalog. HY-15144; catalog. HY-B0150).
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