The expression of ALDH and vimentin in DLD-S ALDH/PI CSCs were analyzed with immunofluorescence staining and the laser confocal technique. For immunofluorescence analysis, cells were grown in serum-free DMEM/F12 (Hyclone; GE Healthcare Life Sciences) medium containing several growth factors and placed onto glass-coverslips for 16 h prior to fixation with 2% paraformaldehyde at 37°C for 15 min. Fixed cells were then permeablized with 0.1% Triton X-100 in PBS at 20°C for 15 min, followed by incubation at 4°C overnight with the following primary antibodies: ALDH (1:200; sc-166362; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and vimentin (1:200; sc-7557; Santa Cruz Biotechnology), respectively. Antibodies were diluted using blocking buffer (PBS containing 2% BSA and 5% FBS). Cells were then incubated with phycoerythrin-(1:200; sc-166362; Santa Cruz Biotechnology, Inc.) or fluorescein isothiocyanate-conjugated secondary antibody (1:200; sc-2024; Santa Cruz Biotechnology, Inc.) at 20°C for 1 h. These slides were then stained using 4′,6-diamidino-2-phenylindole (DAPI; 1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.) at 20°C for 10 min. Fluorescent images were captured using the Olympus FV1200 laser confocal microscope (Olympus Corporation, Tokyo, Japan).
Sc 166362
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-166362 is a laboratory reagent product offered by Santa Cruz Biotechnology. It is a solution intended for use in scientific research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab92742, by Abcam (2 mentions)
Ab57653, by Abcam (2 mentions)
Ab82346, by Abcam (2 mentions)
Nbp2 49415, by Novus Biologicals (2 mentions)
Sab5500117, by Merck Group (2 mentions)
Nbp2 30857, by Novus Biologicals (2 mentions)
Ta305935, by OriGene (2 mentions)
Sc393099, by Santa Cruz Biotechnology (2 mentions)
Immobilon western chemiluminescent hrp substrate kit, by Merck Group (2 mentions)
Ab52865, by Abcam (2 mentions)
Ab75850, by Abcam (2 mentions)
Cd133, by Cell Applications (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Sc 2024, by Santa Cruz Biotechnology (1 mentions)
Sc 7557, by Santa Cruz Biotechnology (1 mentions)
Ab108203, by Abcam (1 mentions)
Sc 20687, by Santa Cruz Biotechnology (1 mentions)
Sc 271786, by Santa Cruz Biotechnology (1 mentions)
Ab38295, by Abcam (1 mentions)
Ab72130, by Abcam (1 mentions)
9 protocols using sc 166362
1
ALDH and Vimentin Expression in DLD-S ALDH/PI CSCs
2
Immunohistochemical Assessment of Tumor Markers
3
Quantifying Liver Protein Expression
4
Determining Protein Expression Levels
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CSC221, cells treated for 48 h were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer24 (link). Antibodies against ALDH1 (sc-166362; Santa Cruz Biotechnology, Dallas, TX, USA), CD133 (CA1217; Cell Applications, San Diego, CA, USA), CD44 (3570; Cell Signaling Technology, Danvers, MA, USA), Lgr-5 (ab75850; Abcam, Cambridge, MA, USA), Msi-1 (ab52865, Abcam), Gli1 (sc-20687; SANTA CRUZ, Dallas, TX, USA), Gli2 (sc-271786; SANTA CRUZ), Smoothened (SMO; ab72130; Abcam, Cambridge, MA, USA), Bmi-1 (ab38295; Abcam) were used to detection. α-tubulin (2125, Cell Signaling Technology) and β-Actin (sc-47778; SANTA CRUZ) antibody was used as an internal control. The bands were cut according to the protein size region of interest before intubating with antibodies and then imaged with an Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore, Billerica, MA, USA). Uncropped images of the blot images are presented as additional data (Supple Figs. 10 , 11 ). Bands relative density was calculated based on the density of α-tubulin and actin bands in each sample. Values were demonstrated as arbitrary densitometric units corresponding to signal intensity.
5
Comprehensive Protein Expression Analysis
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AR(rabbit monoclonal, 1:1000, #5153), PSA/KLK3(rabbit monoclonal, 1:1000, #5365), TOMM20(rabbit monoclonal, 1:1000, #42,406), ATG5(rabbit monoclonal, 1:1000, #12994S), LC3I/II(rabbit monoclonal, 1:1000, #12,741), Akt (rabbit monoclonal, 1:1000, #4691), Phospho-Akt (Ser473)(rabbit monoclonal, 1:1000, #4060), Phospho-Akt (Thr308)(rabbit monoclonal, 1:1000, #13,038), Nanog(rabbit monoclonal, 1:1000, #4903), Sox2(rabbit monoclonal, 1:1000, #3579), ALDH1A1(rabbit monoclonal, 1:1000, #36671S) and VDAC(rabbit monoclonal, 1:1000, #4661) antibodies were purchased from Cell Signaling Technology (CST). NCAM1(rabbit polyclonal antibody, 1:1000, A7913), β-Actin(mouse polyclonal, 1:5000, AC004) and α-Tublin(mouse polyclonal, 1:5000, AC012) antibodies were purchased from ABclonal Technology (Upper Heyford, UK). NSE (rabbit monoclonal, 1:1000, ab180943) and Syp (Rabbit monoclonal, 1:1000, ab184176) were purchased from Abcam (Shanghai, China). ALDH1A1(mouse monoclonal antibody, 1:500,sc-166362), TOMM70(mouse monoclonal antibody, 1:500, sc-390545),AR(rabbit polyclonal, 1:1000, Sc-815X) antibodies were purchased from Santa cruz. E-cadherin (rabbit polyclonal, 1:1000, GTX100443),BRN2(rabbit polyclonal, 1:1000, GTX114650) and N-cadherin (rabbit polyclonal, 1:1000, GTX127345) were purchased from Genetex.
6
Stemness Factor Expression Analysis
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Cells treated with physciosporin for 48 h were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in lysis buffer [30 (link)]. Antibodies against ALDH1 (sc-166362; Santa Cruz Biotechnology, Dallas, TX, USA), CD133 (CA1217; Cell Applications, San Diego, CA, USA), CD44 (3570; Cell Signaling Technology, Danvers, MA, USA), Lgr-5 (ab75850; Abcam, Cambridge, MA, USA), and Msi-1 (ab52865, Abcam) were used to detect stemness factors. α-tubulin (2125, Cell Signaling Technology) antibody was used as an internal control. The luminescence photon from proteins were detected by an Image Quant LAS 4000 mini using horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) with an Immobilon Western Chemiluminescent HRP Substrate Kit (Merck Millipore, Billerica, MA, USA). Quantitation of bands were performed by using Multi-Gauge 3.0 software, and the relative density of each band was calculated based on the density of the internal control bands in each sample. Values are shown as arbitrary densitometric units corresponding to signal intensity. All results are representative of at least three independent experiments.
7
Immunohistochemical Assessment of Tumor Markers
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Immunohistochemical staining and analysis was performed as previously published[30 (link)-32 (link)]. Briefly, Ki67 proliferation indices were determined by counting the number of Ki67 positive tumor cells in 10 individual 40x fields and dividing that number by the total number of tumor cells. The data are expressed as the mean ± S.E.M. of two independent experiments. Expression indices of RRM1, RRM2, hENT1, hCNT1, CDA, dCK, ALDH1, CD133, and CXCR4 were calculated by assigning a staining intensity of 0, 1, 2, or 3 to each specimen, and multiplying this intensity by the percent of tumor cells expressing the protein of interest[33 (link)]. Overall scores ranged from 0 to 300. Data were derived from photomicrographs taken with Olympus BH-2 microscope with DP71 camera and DPS-BSW v3.1 software. Primary antibodies were obtained from multiple sources: Ki67 (ab92742, abcam, Cambridge, MA, USA), RRM1 (NBP2-49415, Novus Biologicals, Littleton, CO, USA), RRM2 (ab57653, abcam), hENT1 (SAB5500117, Sigma-Aldrich, St. Louis, MO, USA), hCNT1 (NBP2-30857, Novus Biologicals), CDA (ab82346, abcam), dCK (sc393099, Santa Cruz, Dallas, TX, USA), ALDH1 (sc166362, Santa Cruz), CD133 (CS#86781T, Cell Signaling, Danvers, MA, USA), CXCR4 (TA305935, Origene, Rockville, MD, USA).
8
Staining of ALDH and Ki-67 Positive Cells
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Cells expressing a high level of ALDHbr and Ki-67 were stained. Briefly, formalin-fixed paraffin-embedded sections of tumor samples were stained with mouse anti-Ki-67 antibody (sc-23900; Santa Cruz Biotechnology, Inc.) and mouse anti-ALDH antibody (sc-166362; Santa Cruz Biotechnology, Inc.) using standard procedures. Immunofluorescent staining was performed as previously described.42 (link)
9
Immunofluorescence and Immunohistochemistry Analysis of Epigenetic Markers
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The cells were fixed in 4% formaldehyde, washed with PBS for 15 min, and permeabilized with 0.2% Triton X-100 for 20 min. After permeabilization, the cells were blocked with bovine serum albumin (BSA) at 37°C for 30 min. Fixed cells were incubated with the antibodies against H3K9me2 (1:200, ab1220; Abcam) at 4°C overnight, followed by Alexa Fluor 594 goat anti-rabbit IgG (H+L) secondary antibody (1:1,000, A-11012; Life Technologies, Gaithersburg, MD, USA) for 1 h at room temperature. The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI, 1:10,000; 4084; Cell Signaling Technology, Inc.). The fluorescence images were obtained using an Olympus microscope.
For immunochemistry test of clinical samples, briefly, formalin-fixed paraffin-embedded samples were prepared as 4-μm-thick sections as previously described (35 (link)). The sections were incubated with primary antibodies against CD133 (ab19898; Abcam) and ALDH1 (sc-166362, Santa Cruz Biotechnology, Inc.) at 4°C overnight, and subjected to be incubated with the appropriate secondary antibody for 30 min at room temperature. IHC staining results in each sample was scored using semiquantitative scoring system, taking into consideration the staining intensity obtained and the proportion of positive cells observed.
For immunochemistry test of clinical samples, briefly, formalin-fixed paraffin-embedded samples were prepared as 4-μm-thick sections as previously described (35 (link)). The sections were incubated with primary antibodies against CD133 (ab19898; Abcam) and ALDH1 (sc-166362, Santa Cruz Biotechnology, Inc.) at 4°C overnight, and subjected to be incubated with the appropriate secondary antibody for 30 min at room temperature. IHC staining results in each sample was scored using semiquantitative scoring system, taking into consideration the staining intensity obtained and the proportion of positive cells observed.
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