The following antibodies were used in this study: primary—mouse anti-E-Cadherin (1:200 610181, BD Transduction Lab), mouse anti-β-actin (1:200, A5441, Sigma), goat anti-Ctr (OBT0978, AbD Serotec), mouse anti-HSP60 (1:5000, ALX-804-701, Alexis), rabbit anti-Ki67 (9027, Cell Signaling), rabbit anti-pSTAT1 (1:1000, 9167, Cell Signaling), rabbit anti-pSTAT3 (Tyr705) (1:1000 9145, Cell Signaling), cleaved caspase-3 (1:1000, 9664, Cell Signaling), goat anti-LIF (1:500, AF-250-NA, R&D Systems); secondary - sheep anti-mouse-HRP (1:3000, NA931, Amersham), donkey anti-rabbit-HRP (1:3000, NA934, Amersham), donkey anti-goat-HRP (1:3000, 800073, Biomol), donkey anti-mouse-Alexa488 (1:300, 715-546-140, Dianova), donkey anti-goat-Cy3 (1:300, 705-165-003, Dianova), donkey anti-rabbit-Alexa488 (1:300, 711-546-152, Dianova), donkey anti-mouse-Dylight 647 (1:300, 715-605-150, Dianova), CD326 (EpCAM)-FITC antibody (1:50, 130-080-301, Miltenyi), mouse anti-human CD24-BV711 antibody (1:200, 563371, BD Biosciences), mouse anti IgG1-APC (1:100, 130-098-846, Miltenyi), and mouse anti-human CD133/1 (AC133)APC (1:100, 130098829, Miltenyi). As a DNA-labeling agent, Draq5 (5 µM, 62252, Thermo Scientific) was used.
Af 250 na
Manufactured by R&D Systems
The AF-250-NA is a laboratory equipment product manufactured by R&D Systems. It serves as a core function for various research applications. The detailed technical specifications and intended use of this product are not available in this factual and unbiased description.
Automatically generated - may contain errors
Lab products found in correlation
A5441, by Merck Group (4 mentions)
Biotinylated secondary antibody, by Vector Laboratories (3 mentions)
Vectastain elite abc kit, by Vector Laboratories (3 mentions)
Amico ultra 4, by Merck Group (2 mentions)
Fl 393, by Santa Cruz Biotechnology (2 mentions)
Ab199, by Abcam (2 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
Mouse anti e cadherin, by BD (1 mentions)
Na931, by Cytiva (1 mentions)
Na934, by Cytiva (1 mentions)
Ab14683, by Abcam (1 mentions)
Sc 1618, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Tween 20, by Bio-Rad (1 mentions)
Sc 10790, by Santa Cruz Biotechnology (1 mentions)
10 protocols using af 250 na
1
Antibody Panel for Cell Characterization
2
Protein Extraction and Western Blot Analysis
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The standard protocol was used to extract proteins and perform Western-blot assays as previously described [37 (link)]. Antibodies against Glut1 (ab14683, Abcam; 1:1,000), anti-LIF (AF-250-NA, R & D; 1:1000), anti-Na/K ATPase (ab58479, Abcam, 1:2,000), anti-p-AKT (4051, Cell Signaling; 1:2,000), anti-AKT (SC-1618, Santa Cruz; 1:2,000), and anti-β-actin (A5441, Sigma; 1:125,000) were used in this study.
3
Immunohistochemical Analysis of LIF and Cleaved Caspase-3
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IHC staining for LIF and Cleaved-caspase 3 was performed as previously described 60 . In brief, tissue sections were deparaffinized in xylene and rehydrated with ethanol. After pre-incubation with 10% normal goat serum in PBS (pH 7.5), tissue sections were incubated with primary antibodies, including anti-LIF (AF-250-NA, R&D, 1:100 dilution) and anti-Cleaved caspase 3 antibodies (D175, cell signaling, 1:1,000 dilution), for overnight at 4°C. Tissue sections were then stained with biotinylated secondary antibody (Vector). Immunoreactivity was detected by using a Vectastain Elite ABC kit (Vector). Known positive controls were included in each experiment, and negative controls were obtained by omitting the primary antibody. The IHC results were scored according to the percentage of cells showing positive staining: -: 0%–<10%; +: ≥10%.
4
Protein Expression Analysis via Western Blot
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Whole tissues were lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM NaF, 25 mM β-glycerolphosphate and protease inhibitor cocktail (Calbiochem) and the protein was quantified by Bradford assay. Equal amounts of protein per sample were resolved on 8–10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) gels, transferred to polyvinyl difluoride (PVDF) membranes (GE Healthcare Bio-Sciences), blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) with 0.1% Tween-20 (Bio-Rad) and probed with polyclonal antibody against LIF (1:500; R&D Systems, #AF-250NA) overnight at 4 °C, followed by three wash steps. Membranes were incubated 1 h at room temperature with secondary anti-rabbit IgG-horse-radish peroxidase (HRP) linked, (1:5000; Dako) and signals were developed with enhanced chemiluminescence Western blotting detection system reagent (Pierce). Membranes were stripped and incubated with anti-GAPDH as a protein loading control.
5
LIF Neutralization in Chlamydia Infection
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For LIF neutralization the general protocol for CtrD infection of organoids (as described above) was carried out. Differing from this protocol, the fragmented organoids were resuspended in 90 µl ADF++, which was added with or without CtrD, and subsequently mixed with 10 µl of 1:40 pre-diluted antibodies. Neutralization was done using 0.5 µg ml−1 anti-LIF antibody (R&D, #AF-250-NA, from goat, 200 µg ml−1) and mock control using 0.5 µg ml−1 goat anti-CagA (Santa Cruz, #sc-6085, 200 µg ml−1). After the specified incubation time for infection, the organoids were seeded in Matrigel and culture medium was added. 14 days p.i. organoids were fixed, embedded and IF labeled for detection of Chlamydia.
6
Western Blot Protein Analysis
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Standard Western-blot assays were used to analyze the levels of protein in cell lysates and CM. CM which was cultured with 1/3 number of the cells used for Western-blot assays was concentrated with an Amico Ultra-4 centrifugal filter device (Millipore) after a brief centrifugation to remove any cell debris. Antibodies against p53 (FL393, Santa Cruz; 1:1,000 dilution), anti-p-p53 (Ser15) (9284, Cell Signaling; 1:1,000 dilution), anti-LIF (AF-250-NA, R&D; 1:1,000 dilution), anti-PARP1/2 (H250, Santa Cruz; 1:1,000 dilution), anti-cleaved-caspase 3 (D175, Cell Signaling; 1:1,000 dilution), anti-MDM2 (2A10; 1:1,000 dilution), anti-p-Stat3 (Tyr705) (9131, Cell Signaling; 1:1,000 dilution), anti-Stat3 (C-20, Santa Cruz; 1:2,000 dilution), anti-ID1 (C-20, Santa Cruz; 1:2,000 dilution), anti-p21 (Ab-6, Calbiochem; 1:1,000 dilution, and anti–β-actin (A5441, Sigma; 1:125,000 dilution) antibodies were used in this study. The full blots are shown in Supplementary Fig. 10 – Supplementary Fig. 14 .
7
Immunohistochemical Analysis of LIF and Cleaved Caspase-3
8
Colorectal Carcinoma Tissue Microarray Analysis
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High-density human colorectal carcinoma tissue microarrays (CO6161) containing duplicated cores for 293 cases of colorectal cancers were purchased from US Biomax. Nine cases with poor core quality were excluded for analysis. IHC staining for LIF and HIF-2α was performed as previously described [17 ]. In brief, tissue sections were deparaffinized in xylene and rehydrated with ethanol. After pre-incubation with 10% normal goat serum in PBS (pH 7.5), tissue sections were incubated with primary antibodies, including anti-LIF (AF-250-NA, R&D, 1:50 dilution) and anti-HIF-2α antibodies (ab199, Abcam, 1:100 dilution), for overnight at 4°C. Tissue sections were then stained with biotinylated secondary antibody (Vector). Immunoreactivity was detected by using a Vectastain Elite ABC kit (Vector). The IHC results were scored according to the percentage of cells showing positive staining: -: 0% to < 10%; +: > 10%. Two cases of colorectal cancer specimens with positive staining of LIF and HIF-2α and two cases of normal adjacent specimens with negative staining of LIF and HIF-2α were included as positive and negative controls, respectively.
9
Quantification of HIF and LIF Proteins
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10
Western Blot Protein Analysis
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