Western blot analysis was performed using specific primary antibodies against CD133 (Proteintech, USA), CD44 (CST, USA), Lgr5 (Abcam, USA), EpCAM (Abcam, USA), ALDH1 (Abcam, USA), β-catenin (Proteintech, USA), Nanog (CST, USA), E-cadherin (CST, USA), N-cadherin (Epitomics, USA), vimentin (CST, USA), Snail (Abcam, USA), Twist (Abcam, USA), Slug (Abcam, USA), ZEB1 (Abcam, USA), fibronectin (Abcam, USA), Sox2 (CST, USA), Oct4 (CST, USA), PRDX2 (Abcam, USA), Cyclin D1 (Abcam, USA), c-Myc (Abcam, USA), MMP-2 (Abcam, USA), MMP-9 (Abcam, USA), VEGF (Abcam, USA) and GAPDH (Goodhere, China).
Aldh1
Manufactured by Abcam
Sourced in United States, United Kingdom
ALDH1 is an enzyme that catalyzes the oxidation of aldehydes to carboxylic acids. It is involved in the metabolism of alcohol and plays a role in the detoxification of acetaldehyde, an intermediate in alcohol metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Nanog, by Abcam (5 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Vimentin, by Abcam (3 mentions)
Abcg2, by Abcam (3 mentions)
N cadherin, by Abcam (3 mentions)
Fibronectin, by Abcam (2 mentions)
Cd133, by Proteintech (2 mentions)
Snail, by Abcam (2 mentions)
Twist, by Abcam (2 mentions)
Epcam, by Abcam (2 mentions)
β catenin, by Proteintech (2 mentions)
Cyclin d1, by Abcam (2 mentions)
β catenin, by BD (2 mentions)
Vimentin, by BD (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Nanog, by Cell Signaling Technology (2 mentions)
β actin, by Abcam (2 mentions)
E cadherin, by Abcam (2 mentions)
Prdx2, by Abcam (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
19 protocols using aldh1
1
Comprehensive Stem Cell Marker Analysis
2
Comprehensive Western Blot Analysis of Stem Cell Markers
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Cells were lysed in lysis buffer (1% NP-40, 50 mM Tris, 0.1% SDS, 1 mM PMSF, 10 mM EDTA, 150 mM NaCl, and 0.5% sodium deoxycholate) as instructed (Beyotime, China). Supernatants of lysates were collected after centrifugation. A BCA protein detection kit (Beyotime, China) was used to determine the protein concentration. SDS/PAGE was used for the separation of the indicated amounts and then transferred on to PVDF membranes (Millipore, U.S.A.). Using nonfat milk, the membranes were blocked for 1 h. Next, primary antibodies were incubated overnight at 4°C. Then, the membranes were washed using TBST for 15 min and incubated with secondary antibodies (1:5000) at 37°C for 1 h. After washing with TBST for 15 min, the detection was performed with Fusion FX (Vilber, France) using an enhanced chemiluminescence kit (Millipore, U.S.A.). Specific bands were quantified using Fusion software. Each experiment was performed in triplicate. The following antibodies were used: CD133 (Proteintech, U.S.A.), CD44 (CST, U.S.A.), Lgr5 (Abcam, U.S.A.), EpCAM (Abcam, U.S.A.), ALDH1 (Abcam, U.S.A.), β-catenin (Proteintech, U.S.A.), CD166 (CST, U.S.A.), E-cadherin (CST, U.S.A.), N-cadherin (Epitomics, U.S.A.), vimentin (CST, U.S.A.), Snail (Abcam, U.S.A.), Twist (Abcam, U.S.A.), Slug (Abcam, U.S.A.), ZEB1 (Abcam, U.S.A.), fibronectin (Abcam, U.S.A.), and GAPDH (Goodhere, China).
3
Western Blot Analysis of EMT and Stemness Markers
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Cell lysates were harvested in ice-cold modified radioimmune precipitation assay buffer containing a protease inhibitor cocktailTM (Roche). Lysates normalized for amount protein were separated on 10% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose. The blots were incubated with primary antibodies to E-CADHERIN, VIMENTIN, FIBRONECTIN, β-CATENIN (BD), c-MYC, FOXM1, TFIIB, MYD88, γ-CATENIN, SNAIL1, SLUG, TWIST, hCTR1 (Santa Cruz), BMI1, CD44, NANOG, ALDH-1, OCT-4, NOTCH-1, SFRP5 (Abcam), ZO-1 (Invitrogen), phospho-β-CATENIN, N-CADHERIN (Cell Signaling), or SOX-2 (Sigma). Immunocomplexes were detected with horseradish peroxidase-conjugated IgG and visualized via enhanced chemiluminescence (ECL detection kit, Amersham).
4
Western Blot Analysis of Stem Cell Markers
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CRC tissues and cell lines were collected and lysed in radioimmunoprecipitation assay buffer with PMSF (Beyotime, Shanghai, China). Protein samples were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred in the polyvinylidene difluoride membranes (Millipore). Then, 5% non-fat milk was used to block the membranes for 2 h. Subsequently, the membranes were incubated with primary antibodies at 4°C overnight and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody. Each band was visualized by enhanced chemiluminescence reagents (Yeasen, Shanghai, China). The following antibodies were used for Western blotting: HIST2H2BF (Thermo Fisher Scientific, USA, 1:1000), NICD (CST, Beverly, MA, USA, 1:1000), Hes1 (CST, 1:1000), Hey1 (Abcam, 1:1000), CD133 (Abcam, 1:1000), CD44 (Abcam, 1:1000), ABCG2 (Abcam, 1:1000), ALDH1 (Abcam, 1:1000), Nanog (Abcam, 1:1000), Bmi-1 (Abcam, 1:1000), Oct-4 (Abcam, 1:1000), glyceraldehyde-3-phosphate dehydrogenase (Abcam, 1:1000), horseradish peroxidase-linked anti-rabbit IgG (CST, 1:3000), and horseradish peroxidase-linked anti-mouse IgG (CST, 1:3000). glyceraldehyde-3-phosphate dehydrogenase was used as an internal control.
5
Immunohistochemical Analysis of Tumor Samples
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The paraffin embedded tumor and normal tissue sections were stained using Ki67 (1:200, Cell signaling) and ALDH1 (1:100, Abcam) antibodies then stained with biotinylated anti-mouse immunoglobulin G (H + L) secondary antibody followed by incubation in ABC reagent (DAKO Labs, Cambridgeshire, UK). For H&E staining, the slides were deparaffinized, rehydrated and stained with hematoxylin and eosin for 1 minute. The slide was mounted with 3,3'-diaminobenzidine and visualized under a light microscope.
6
Immunohistochemical Profiling of Cancer Markers
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The synthesis of NCT-58 has been described previously [29 (link)]. Primary antibodies targeted Ki-67, CD31, ALDH1 and CD44 (Abcam, MA); HER2, phospho-HER2 (Tyr1221/1222), HER3, phospho-HER3 (Tyr1289), EGFR, phospho-EGFR, Akt, phospho-Akt (Ser473), PARP, cleaved-PARP, cleaved-caspase-3, cleaved-caspase-7, vimentin, Nanog, Oct4, Sox2, Ras, Raf (Ser338), phospho-Raf, Mek, phospho-Mek (Ser217/221), Erk and phospho-Erk (Tyr202/204) (Cell Signaling, CA); CB11 (Thermo Fisher Scientific Fremont, CA); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems, AZ); survivin, HSP70, HSP90, and HSF-1 (Santa Cruz Biotechnology, CA); and GAPDH (Invitrogen, CA). Secondary antibodies were HRP-conjugated anti-rabbit and mouse IgG (Bio-Rad Laboratories, CA) and Alexa Fluor-488 and −594 goat anti-rabbit IgG (Invitrogen).
7
Immunocytochemical Characterization of Stem Cells
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Cell suspensions were seeded on the central concavity of a special 35 mm glass bottom plate (Nest, China) at ~ 50% confluent density. The cells were cultured overnight with or without PLA. After three washes with ice-cold PBS, cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 for 10 min. Subsequently, cells were blocked with 5% bovine serum albumin for 1 h at room temperature and incubated with an appropriate concentration of primary antibodies against Oct4, ABCG2, ALDH1, SOX2, or CD49f (Abcam, London, UK, 1:100 dilution) overnight at 4 °C. Then, the cells were incubated with Alexa Fluor® 488 conjugated goat anti-rabbit IgG (Abcam, London, UK, 1:200 dilution) or goat anti-mouse IgG (Abcam, London, UK, 1:200 dilution) secondary antibodies for 1 h. The stained cells were observed under a fluorescence confocal microscope.
8
Immunofluorescence Assay for Premalignant Tumors
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The immunofluorescence experiment, Figure 5 , was conducted using a minimum of 15 premalignant tumors from each treatment group, following the previously described protocol [5 (link)]. Briefly, deparaffinized slides were covered with the primary antibody solution. The primary antibodies used included β-catenin (Cat# SC-7963, Santa Cruz Biotechnology, Dallas, TX), BMI1 (Cat#NBP187321, Novus Biologicals), and ALDH1 (Cat# ab23375 Abcam), each diluted at 1:100. The slides were incubated with the primary antibodies overnight at 4 °C. The slides were taken out and washed three times in PBST. Appropriate fluorescence-labeled secondary antibodies were then applied for 1 h at room temperature (Alexa Flour 488 donkey anti-rabbit IgG (H + L) Cat#1796375, and Alexa Flour 594 donkey anti-mouse IgG (H + L) Cat#2069656, Invitrogen Life Technologies, Carlsbad, CA, USA). The slides were then mounted using a mounting medium containing DAPI for imaging.
9
Immunohistochemical Analysis of FFPE Tissue
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FFPE tissue sections (4-μm thick) were used for immunohistochemistry. Deparaffinization was followed by heat-based antigen retrieval, endogenous peroxidase blockade with 3% hydrogen peroxide, and blocking with normal sera. The primary antibodies used were as follows: MCM2, mouse monoclonal, 1:2000 (BD Biosciences, San Jose, CA, USA); CD133, mouse monoclonal, 1:100 (Miltenyi Biotec, Auburn, CA, USA); ALDH-1, rabbit monoclonal, 1:1000 (Abcam, Cambridge, UK); FLAG, mouse monoclonal, 1:250 (InvivoGen, San Diego, CA, USA); and GFP, mouse monoclonal, 1:100 (Abcam). The primary antibodies were incubated overnight at 4°C. Primary antibodies were detected using an ABC Kit (Vector Laboratories, Burlingame, CA, USA) or EnVision+ System-HRP (Dako, Glostrup, Denmark) with diaminobenzidine (DAB; Nichirei Bioscience, Japan) or the HISTOFINE simple stain AP series (Nichirei Bioscience) with Vector Blue (Vector Laboratories). A TUNEL assay was also performed using the In Situ Cell Death Detection Kit, POD (Roche Diagnostics, Tokyo, Japan) with DAB. For double immunostaining, samples were heat treated and blocked between each step.
10
Immunohistochemical Analysis of Stem Cell Markers
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The immunohistochemistry analysis was performed as described below. After deparaffinisation and rehydration, the TMA sections were subjected to high pressure for 2 min to achieve antigenic retrieval. The slides were incubated overnight at 4°C with the following primary antibodies: Hes1 (1:500 dilution; Bioss), ALDH1 (1:500 dilution; Abcam), and Bmi-1 (1:500 dilution; Abcam). The sections were then incubated with DAB for 2 min. In every run, the primary antibodies were substituted with PBS for the negative controls.
For the evaluation of the IHC results, the proportion of stained tumour cells was evaluated using four grades: 0, no positive tumour cells; 1, <10% positive tumour cells; 2, 10–50% positive tumour cells; and 3, >50% positive tumour cells. Similarly, the scoring criteria for the staining intensity were the following: 0, no staining; 1, weak staining; 2, modest staining; and 3, strong staining. The final score was calculated by multiplying the tumour staining area by the intensity score (0, 1, 2, 3, 4, 6, and 9). According to this method of assessment, staining scores ≤4 and ≥6 were regarded as tumours with low and high expression, respectively.
For the evaluation of the IHC results, the proportion of stained tumour cells was evaluated using four grades: 0, no positive tumour cells; 1, <10% positive tumour cells; 2, 10–50% positive tumour cells; and 3, >50% positive tumour cells. Similarly, the scoring criteria for the staining intensity were the following: 0, no staining; 1, weak staining; 2, modest staining; and 3, strong staining. The final score was calculated by multiplying the tumour staining area by the intensity score (0, 1, 2, 3, 4, 6, and 9). According to this method of assessment, staining scores ≤4 and ≥6 were regarded as tumours with low and high expression, respectively.
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