Slides containing human CRC tissue sections were obtained from the Department of Pathology of Harbin Medical University. IHC analysis of CRC tissue sections on slides was performed using 20 μg/mL anti-DCLK1 (DCLK1-87) and anti-ALDH1 mAbs (1:250, ab134188; Abcam). A rabbit IgG antibody was used as a negative control (1:250, A7016; Biyuntian, Nanjing, People’s Republic of China). Immunostaining was performed on 5-μm slices of formalin-fixed paraffin-embedded tumor xenografts. Antigen retrieval was performed by microwave heating, and nonspecific protein-binding sites were blocked with 4% normal goat serum and 1% BSA in PBS for 1 hour. The 5-μm sections were incubated with anti-DCLK1 (DCLK1-87) and anti-ALDH1 mAbs (ab134188; Abcam). In parallel, nonimmune rabbit IgG was used as a negative control. A biotinylated secondary antibody, an avidinbiotinylated enzyme complex, and a 3,3′-diaminobenzidine substrate were used for detection (ZLI-9018; Zhongshan Golden Bridge Biotechnology). The slides were counterstained with hematoxylin and mounted with mounting medium for analysis. Images were captured using a Leica DM4000B microscope (Leica Microsystems, Wetzlar, Germany).
Ab134188
Manufactured by Abcam
Sourced in United Kingdom
Ab134188 is a lab equipment product manufactured by Abcam. It is a versatile tool designed for scientific research applications. The core function of this product is to facilitate experimental procedures in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Zli 9018, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Dm4000b microscope, by Leica (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Ab157107, by Abcam (1 mentions)
Ab14592, by Abcam (1 mentions)
A2228, by Merck Group (1 mentions)
Chemiluminescence detection system, by ATTO Corporation (1 mentions)
Ab37994, by Abcam (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Ab109250, by Abcam (1 mentions)
Ab181557, by Abcam (1 mentions)
Ab109244, by Abcam (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Ab215036, by Abcam (1 mentions)
Ab223582, by Abcam (1 mentions)
Skl2001, by Merck Group (1 mentions)
6 protocols using ab134188
1
Immunohistochemical Analysis of CRC Tissues
2
Immunohistochemistry Protocol for ALDH1A1
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Before dewaxing, the tissue sections were placed in a 60°C baking oven for 20 minutes. Slides were deparaffinized in xylene, rehydrated in a graded alcohol series, and washed in PBS twice for five minutes each time. Sections were heated in 10 mM sodium citrate buffer, pH = 6.0, for 15 minutes in a 95°C water bath for antigen retrieval. Until the buffer cooled down, we performed five-minute PBS washes. Endogenous peroxidase activity was blocked by incubating the sections in 3% H2O2 at room temperature for ten minutes. Blocking serum was added dropwise at room temperature for 20 minutes to reduce the non-specific background. Anti-ALDH1A1 monoclonal antibodies (ab-134188; 1:100 dilution; Abcam, Cambridge, MA, USA) were added and incubated overnight at 4°C. The sections were washed in PBS three times for two minutes, and subsequently incubated with biotinylated secondary antibody (PK-4001; VECTASTAIN® Elite ABC kits, Vector Labs, USA) for 30 minutes at room temperature. The slides were subsequently incubated with ABC (PK-4001; VECTASTAIN® Elite ABC kits, Vector Labs, USA) for another 30 minutes, washed in PBS, and stained with DAB (3, 3-diaminobenzidine). Finally, the sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted.
3
Protein Isolation and Western Blotting
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Protein isolation and western blotting were done as previously reported (26 (link)). Each protein band was normalized against β-actin. Anti-NEDD4 antibody (ab14592, Abcam; Cambridge, Cambridgeshire, United Kingdom) was used at 10,000:1 dilution, while anti-ALDH1A1 (ab134188) and anti-CD44 antibodies (ab157107, Abcam; Cambridge, Cambridgeshire, United Kingdom) as well as anti-β-actin antibody (A2228, Sigma Aldrich; St. Louis, MO, United States) were used at 1,000:1 dilution. The membrane was developed using a chemiluminescence detection system (ATTO Corporation, Tokyo, Japan).
4
Comparative Analysis of Colon Cell Lines
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NCM460 and HCT116 cell lines were purchased from American Type Culture Collection. NCM460 cells are a type of normal colon cell, and HCT116 is a human colon cancer cell line. Both cell types were maintained in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. We used a polyclonal rabbit anti-DCLK1 antibody (ab37994; Abcam, Cambridge, UK) and a rabbit anti-ALDH1 mAb (ab134188; Abcam) as positive controls.
5
Characterization of Breast Cancer Cell Lines
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MCF10A, BT549, and 4T1 cells were purchased from Beijing union medical college hospital cell resource-sharing platform. SUM159 cells were a gift from Dr. Jianjun He. MCF10A was grown in DMEM/F12 media supplemented with 5% horse serum, 20 ng/ml human EGF, 10 μg/ml insulin, 0.5 μg/ml hydrocortisone, 1% penicillin and streptomycin. BT549 was cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS), 1 μg/ml insulin, and 1% penicillin and streptomycin. SUM159 was cultured in DMEM/F12 medium with 10% FBS and 1% penicillin and streptomycin. 4T1 was cultured in RPMI 1640 medium with 10% FBS and 1% penicillin and streptomycin. Medium, FBS and Penicillin-Streptomycin were purchased from Corning. All these cell lines were maintained in a humidified incubator at 37°C, 5% CO2. Antibodies against RICH1 was purchased from Santa Cruz (sc-514438). Antibodies against Merlin (ab109244), ALDH1A1 (ab134188), Nanog (ab109250), Oct4 (ab181557), E-Cadherin (ab40772) were purchased from Abcam. Antibodies against LATS1 (#3477), phospho-LATS1T1079 (#8654), YAP (#14074), phosphor-YAPS127(#13008), TAZ (#70148), phosphor-TAZS89(#59971),CD44 (#3570) were purchased from Cell Signaling Technology. The antibody against Amot was synthesized by Genemed Synthesis Inc. HRP-labeled GAPDH (HRP-60004) and antibody against lamin A/C (10298-1-AP) were from Proteintech.
6
Antibody Profiling for Stem Cell Markers
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The following antibodies were used: SNTB1 (Abcam, ab242046, 1:2,000), α-tubulin (CST, #3873s, 1:2,000), CCND1 (CST, #55506, 1:1,000), c-Myc (CST, #18583, 1:1,000), β-catenin (CST, #8480, 1:1,000), CD133 (CST, #64326, 1:2,000), CD44 (CST, #37259, 1:2,000), EpCAM (Abcam, ab223582, 1:2,000), KLF4 (Abcam, ab215036, 1:2,000), ALDH1A1 (Abcam, ab134188, 1:2,000), POU5F1 (Abcam, ab230429, 1:2,000), and LGR5 (Abcam, ab273092, 1:2,000). The β-catenin agonist SKL2001 (Sigma, USA) was dissolved in dimethylsulfoxide (DMSO) and stored at –20 °C.
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