Cells were lysed in cell lysate, and then centrifuged at 12,000×g for 20 min at 4 °C. The supernatant was collected and denatured. Proteins were separated in 10% SDS-PAGE and blotted onto polyvinylidene difluoride membrane (PVDF). The PVDF membrane was treated with TBST containing 50 g/L skimmed milk at room temperature for 4 h, followed by incubation with the primary antibodies against the LC3B (1:3000, ab51520, Abcam), Atg5 (1:1000, ab108327, Abcam), Atg7 (1:100,000, ab524721,Abcam), Beclin1 (1 μg/ml, ab62557, Abcam), P62 (1:500, 18,470–1-AP, Proteintech, China), OCT-4 (1:1000, Proteintech), ABCG2 (1:50, ab24115, Abcam), c-Myc (1:10,000, ab32072, Abcam), Sox2 (1 μg/ml, ab97959, Abcam) and anti-β-actin (1:1000, Cell signaling) respectively, at 37 °C for 1 h. Membranes were rinsed and incubated for 1 h with the correspondent peroxidase-conjugated secondary antibodies. Chemiluminescent detection was performed with the ECL kit (Pierce Chemical, Rockford, IL, USA). The amount of the protein of interest, expressed as arbitrary densitometric units, was normalized to the densitometric units of ß-actin.
Ab24115
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab24115 is a laboratory equipment product from Abcam. It is designed for a specific function within the laboratory setting. No further details can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Hpa001637, by Merck Group (2 mentions)
Ab108327, by Abcam (1 mentions)
Ab97959, by Abcam (1 mentions)
Ab62557, by Abcam (1 mentions)
Ab51520, by Abcam (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Ab32072, by Abcam (1 mentions)
Ab194356, by Abcam (1 mentions)
Nbp1 44090, by Novus Biologicals (1 mentions)
Hpa002199, by Merck Group (1 mentions)
Af806, by R&D Systems (1 mentions)
Aperio at2 system, by Leica (1 mentions)
Ab2356, by Abcam (1 mentions)
Ab45939, by Abcam (1 mentions)
Ab12290, by Abcam (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab1416, by Abcam (1 mentions)
Ab103477, by Abcam (1 mentions)
Ab109536, by Abcam (1 mentions)
Ab24102, by Abcam (1 mentions)
11 protocols using ab24115
1
Protein Expression Analysis by Western Blotting
2
Immunohistochemical Profiling of Vascular Markers
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IHC was performed on 6 μm sections of formalin-fixed, paraffin-embedded tissues. The sections were deparaffinized and dehydrated, and antigen retrieval followed. Then, the sections incubated with primary antibody toward CD31 (AF806, R&D Systems), CAVIN2 (NBP1-44090, Novus), HSPG2 (AF2364, R&D Systems), MYO1B (ab194356, Abcam), ABCG2 (ab24115, Abcam), SLC2A1 (HPA058494, Sigma-Aldrich), ABCB1 (HPA002199, Sigma-Aldrich), TJP1 (HPA001637, Sigma-Aldrich), CLDN5 (341600, Invitrogen), and PLVAP (NBP1-83911, Novus). All quantification was done by 2 individual scientists in a blinded fashion.
3
Quantitative Immunohistochemical Analysis of Brain Tumor Vasculature
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Tumor-bearing mouse brains were fixed in 4% (v/v) formaldehyde, paraffin embedded and cut into 4 μm coronal sections that were stained with hematoxylin and eosin (H&E), and for P-gp (1:200; 13978; Cell Signaling Technology; Danvers, MA) and BCRP (1:400; ab24115; Abcam; Cambridge, UK). Sections were scanned and processed using an Aperio AT2 system and ImageScope software v12 (both Leica; Wetzlar, Germany).
4
Western Blot Analysis of Lung Cancer Cells
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Human lung cancer cells were transfected with the relevant
plasmids and cultured for 36 h. For western blot analysis, cells were lysed
in NP-40 buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA
pH 8.0, 1 mM EGTA pH 8.0, 1 mM PMSF, and 0.5% NP-40) at 25 °C for 40 min.
The lysates were added to 5× loading dye and then separated by
electrophoresis. The primary antibodies used in this study were 1:1000
rabbit anti-Flag (sc-166,384, Santa Cruz, Dallas, TX, USA), 1:1000 Abcam
(Cambridge, UK) antibody of UBE2C (ab12290), ABCG2 (ab24115), ERCC1
(ab2356), Vimentin (ab45939), E-cadherin (ab1416), cleaved caspase-3
(ab32042) and Tubulin (ab6046).
plasmids and cultured for 36 h. For western blot analysis, cells were lysed
in NP-40 buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA
pH 8.0, 1 mM EGTA pH 8.0, 1 mM PMSF, and 0.5% NP-40) at 25 °C for 40 min.
The lysates were added to 5× loading dye and then separated by
electrophoresis. The primary antibodies used in this study were 1:1000
rabbit anti-Flag (sc-166,384, Santa Cruz, Dallas, TX, USA), 1:1000 Abcam
(Cambridge, UK) antibody of UBE2C (ab12290), ABCG2 (ab24115), ERCC1
(ab2356), Vimentin (ab45939), E-cadherin (ab1416), cleaved caspase-3
(ab32042) and Tubulin (ab6046).
5
Protein Expression Analysis by Western Blot
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Proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, A, USA). After blocking in 5% nonfat milk for 1 h at room temperature, the membranes were incubated overnight with primary antibodies against ID4 (Cat.# ab49261, Abcam, USA), Notch1 (Cat.# ab52627, Abcam, USA), CBF1 (Cat.# ab180588, Abcam, USA), Hes1 (Cat.# 108937, Abcam, USA), JAG1 (Cat.#ab109536, Abcam, USA), MRP1 (Cat.# ab24102, Abcam, USA), ABCG2 (Cat.# ab24115, Abcam, USA), P-gp (Cat.#ab103477, Abcam, USA) and GAPDH (Cat.#ab8245, Abcam, USA) at 4℃. Then, the membranes were incubated for 1 h at 4℃ with the appropriate HRP-conjugated secondary antibodies (Cat. #7074, Cell Signaling, Beverly, MA, USA). Protein expression levels were detected via enzyme-linked chemiluminescence (Pierce, Rockford, USA).
6
Histological Analysis of Mouse Brain
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Whole mouse heads were fixed overnight in a solution containing 4% (v/v) PFA and 5% (v/v) glacial acetic acid. Samples were subsequently decalcified using a 6.5% (v/v) formic acid solution for 4 days at 37°C. Decalcified tissues were paraffin embedded and cut into 4 µm coronal sections that were stained with hematoxylin and eosin, and for SMARCB1 (1:100; 612111, BD Biosciences), MELK (1:50; ab129373, Abcam), Ki-67 (1:3000; ab15580, Abcam), glial fibrillary acidic protein (GFAP) (1:500; BT46-5002–04, BioTrend), S100 (1:1000; Z0311, DakoCytomation), human vimentin (1:4000; M0725, DakoCytomation), cytokeratin-5/6 (1:100; M7237, DakoCytomation), breast cancer resistance protein (BCRP) (1:1000; ab24115, Abcam), permeability glycoprotein (P-gP) (1:1000, #13978, Cell Signaling Technologies), claudin 5 (CLDN5) (1:50; 34–1600, Invitrogen), and glucose transporter 1 (GLUT1) (1:100–2000; 07-1401, Millipore). Tumor and normal brain tissue from the patient were collected in 4% (v/v) PFA and processed and stained for GLUT1 and CLDN5 as previously described.24 (link)
7
Immunohistochemical Analysis of Ovarian BCRP and MDR-1
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Whole ovaries were fixed in DietricH’s Solution and then embedded in paraffin blocks. Five-micron sections of these tissues then were rehydrated through a xylene, ethanol series and then rinsed with PBS. Heat-induced epitope retrieval was performed using sodium citrate buffer. Endogenous peroxidase activity was inhibited with a 3% aqueous peroxide solution and then blocked with 10% fetal calf serum 0.1% Tween-20 1% BSA in PBS for 1 h. The antibodies used were rat monoclonal ab24115 (Abcam Cambridge, UK) for BCRP localization and for MDR-1 sC1517 goat polyclonal from (Santa Cruz Biotechnology, Dallas, TX).
8
Immunohistochemical Staining Protocol
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Immunohistochemical reactions were performed according to standard laboratory methods [84 (link)]. In brief, heat-induced antigen retrieval was performed after dewaxing and rehydration, followed by blocking of endogenous peroxidase with 3% H2O2 in methanol. Incubation with the primary antibodies BCRP (Abcam; ab24115) and CD31 (Abcam; ab28364) was overnight. Subsequently, CD31 sections were conjugated with Labelled Polymer-HRPAnti-Rabbit Envision (DakoCytomation; K4005) while conjugation of the BCRP sections was performed with Goat-α-Rat-Bio (Santa Cruz; SC-2041) and Streptavidin/HRP (DakoCytomation; P0397) respectively. Visualization was carried out with a diaminobenzidine solution. All washing procedures were conducted in phosphate-buffered saline. Slides were counterstained with haematoxylin.
9
Immunohistochemical Analysis of Vascular Markers
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Tissue sections of formalin-fixed, paraffin-embedded samples were deparaffinized and dehydrated prior to antigen retrieval followed by blocking as previously described (19 (link)). Then the sections were incubated with primary antibody towards PLVAP (HPA002279, Sigma), ABCG2 (ab24115, Abcam), and TJP1 (HPA001637, Sigma) followed by incubation with biotinylated secondary antibody and streptavidin conjugated to HRP (Vector Laboratories). The staining was developed with the DAB substrate kit (SK-4100, Vector Laboratories) according to the manufacturer’s protocol.
The images of immunohistochemical staining for VWF, CLDN5, CDH5, PECAM1, and ELTD1 in tumor were obtained from The Human Protein Atlas database (https://www.proteinatlas.org/ ).
The images of immunohistochemical staining for VWF, CLDN5, CDH5, PECAM1, and ELTD1 in tumor were obtained from The Human Protein Atlas database (
10
Immunohistochemical Analysis of Murine Skin
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Murine dorsal skins were embedded in OCT compound (Tissue-Tek; American Master Tech Scientific), frozen, and sectioned following standard protocols. Sections were blocked with 10% normal serum, and stained with antibodies for anti-CD34 (BD Biosciences, Pharmingen, San Jose, CA), anti-BCRP1/ABCG2 (ab24115, Abcam, Cambridge, MA) and anti-MTS24/Plet-1 (sc-240781, Santa Cruz Biotech.) followed by incubation with Alexafluor secondary antibodies (FITC re-conjugated anti-Rat or anti-goat; ThermoFisher, Molecular probes, Waltham, MA). Frozen cross-sections were counterstained with 4′6′-diamidino-2-phenylindole (DAPI) and visualized under a fluorescence microscope using a 465 to 495 nm filter. Paraffin-embedded sections from mouse skin papillomas were immunostained with anti-MTS24 and anti-ABCG2 antibodies described above. The number of MTS24+ and ABCG2+ cells were determined in sections of 250 µm2 with a reticular grid.
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