The excised subcutaneous tumors were fixed in 4% paraformaldehyde for 24 hours at 4°C, and paraffin sections were prepared. Immunohistochemical staining was performed using Histofine Simple Stain MAX PO (Nichirei biosciences, Tokyo, Japan) and ImmPACT DAB peroxidase substrate (Vector Laboratories, Burlingame, CA, USA) on deparaffinised sections treated with 10 mM Tris-HCl (pH 10.0). The anti-Sox2 (Cell Signaling Technology Inc.) and anti-Survivin antibodies (Cell Signaling Technology Inc.) were used as primary antibodies. Representative images were taken using a BZ-X800 microscope (KEYENCE, Osaka, Japan).
Anti survivin antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-survivin antibody is a laboratory reagent used for the detection and quantification of the survivin protein. Survivin is an inhibitor of apoptosis protein that plays a role in cell division and survival. The antibody can be used in various immunoassay techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to study the expression and localization of survivin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Protein plus agarose, by Santa Cruz Biotechnology (2 mentions)
Horseradish peroxidase conjugated anti ub, by Enzo Life Sciences (2 mentions)
N ethylmaleimide, by Merck Group (2 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Anti sox2, by Cell Signaling Technology (1 mentions)
Histofine simple stain max po, by Nichirei Biosciences (1 mentions)
Immpact dab peroxidase substrate, by Vector Laboratories (1 mentions)
Bz x800 microscope, by Keyence (1 mentions)
Anti cyclin b, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti myc, by Santa Cruz Biotechnology (1 mentions)
Anti phospho s10 h3, by Cell Signaling Technology (1 mentions)
Ym 155, by Chemietek (1 mentions)
Alamar blue, by Thermo Fisher Scientific (1 mentions)
Des 1 3 igf 1, by Cell Sciences (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Lapatinib, by LC Laboratories (1 mentions)
Gemcitabine, by Merck Group (1 mentions)
11 protocols using anti survivin antibody
1
Immunohistochemical Analysis of Tumor Samples
2
YAP Protein Immunoprecipitation and Western Blotting
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The YAP antibodies from Abnova (H00010413-M01) and Abcam (52771) were used for immunoprecipitation of endogenous YAP and for Western blotting, respectively, throughout the study. Rabbit polyclonal phospho-specific antibody against YAP T119 has been previously described [14 (link)]. Anti-β-actin, anti-Myc, and anti-cyclin B antibodies were from Santa Cruz Biotechnology. Anti-phospho-S10 H3 and anti-survivin antibodies were from Cell Signaling Technology. Immunoprecipitation and Western blotting assays were done as previously described [37 (link)].
3
Evaluating (des[1-3]IGF-1) Cytotoxicity
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des[1-3]IGF-1 (desIGF1), an N-terminally truncated form of insulin-like growth factor-1, was obtained from Cell Sciences (Canton, MA). AlamarBlue, Dulbecco Modified Eagle Medium (DMEM)/F-12 culture medium and fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA). YM-155 was obtained from Chemietek (Indianapolis, IN) and lapatinib from LC Laboratories (Woburn, MA). Anti-β-actin and anti-survivin antibodies were obtained from Cell Signaling Technology (Beverly, MA). lapatinib was dissolved in dimethyl sulfoxide (DMSO) and diluted as appropriate in water. DMSO concentration did not exceed 0.1% in tissue culture experiments; at this maximal concentration, DMSO alone had no impact on cell number as assessed by AlamarBlue. All experiments involving lapatinib were controlled using treatment with vehicle which contained an equivalent concentration of DMSO.
4
Survivin Protein Ubiquitination Assay
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Experimental methods were described in a previous study [38 (link)]. In brief, the sonicated cell lysates in RIPA lysis buffer containing 10 mM N-ethylmaleimide (Sigma-Aldrich) and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich) were incubated overnight with anti-survivin antibody (Cell Signaling Technology) and subsequently with protein PLUS-Agarose (Santa Cruz Biotechnology) for 2 h at 4 °C. Immunoprecipitation assay was performed with Western blotting using specific primary antibodies, and ubiquitination assay was detected using horseradish peroxidase-conjugated anti-Ub (Enzo Life Sciences) under denaturation conditions.
5
Evaluating Cellular Stress Responses
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Anti-β-actin antibody (A1978) was purchased from Sigma-Aldrich (St. Louis, MO, USA), and anti-nuclear factor erythroid 2-related factor 2 (NRF2) antibody (#12721), anti-glucocorticoid receptor (GR) antibody (#12401), anti-phospho-c-Jun antibody (#9261), anti-c-Jun antibody (#9165), and anti-survivin antibody (#2808) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Dexamethasone (Fuji Pharma Co., Ltd., Tokyo, Japan) was dissolved in phosphate-buffered saline (PBS) to prepare a 1 mM stock solution. 5-FU, gemcitabine, 2′,7′-dichlorofluorescein diacetate (DCF-DA), N-acetyl-cysteine (NAC), and brusatol were purchased from Sigma-Aldrich, and SP600125 was purchased from Merck Millipore (Darmstadt, Germany). They were dissolved in dimethyl sulfoxide (DMSO) to prepare 200 mM 5-FU, 1 mM gemcitabine, 20 mM DCF-DA, 5 M NAC, and 50 mM brusatol stock solutions.
6
Immunohistochemical Analysis of Paraffin-Embedded Tissue
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Preparation of paraffin-embedded tissue sections and immunohistochemical analyses were done as previously described [70 (link)]. Rabbit polyclonal antibodies against AR (Affinity BioReagents, Golden, CO), PSA (Dako, Carpinteria, CA), and rabbit monoclonal anti-survivin antibody (#2808, Cell Signaling Technologies, Beverly, MA) were used. Biotinylated anti-rabbit IgGs and peroxidase-linked avidin/biotin complex reagents were obtained from Vector Laboratories (Burlingame, CA). Control sections were processed in parallel with rabbit nonimmune IgG (Dako, Carpinteria, CA) used at the same concentrations as the primary antibodies.
7
Western Blot Analysis of Protein Targets
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Cells were lysed with RIPA Lysis Buffer (P0013B, Beyotime, China) or Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime, China). Then protein concentrations were quantified with BCA Protein Assay Kit (P0011, Beyotime, China). Twenty microlitre of proteins were separated by SDS-PAGE and then transferred onto PVDF membranes. After blocking, the membranes were incubated with the one of following antibodies: anti-CARMA3 antibody (1:1000, bs-7081R, Bioss, China), anti-MMP2 antibody (bs-0412R, Bioss), anti-MMP9 antibody (1:1000, bs-4593R, Bioss, China), anti-β-catenin antibody (1:1000, #8480, Cell Signaling Technology, USA), anti-Survivin antibody (1:1000, #2808, Cell Signaling Technology, USA), anti-C-myc antibody (1:500, sc-40, Santa Cruz Biotechnology, USA), anti-beta-Actin (1:5000, bsm-33036 M, Bioss, China), or anti-Histone H3.1 (1:500, bs-17422R, Bioss, China) at 4 °C overnight. Then the membranes were incubated in HRP-labeled Goat Anti-Rabbit IgG (H + L) (1:5000, A0208, Beyotime, China) or HRP-labeled Goat Anti-Mouse IgG (H + L) (1:5000, A0216, Beyotime, China) at 37 °C for 45 mins. After incubating ECL reagent for 5 mins, the proteins were detected by ECL Select Western Blotting Detection System (Beyotime, China).
8
Immunoblotting and Immunofluorescence Analysis
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VP (cat. no. SML0534) was purchased from Sigma-Aldrich; Merck KGaA. The following primary antisera were used for the immunoblotting analysis: Anti-β-actin antibody (cat. no. 13E5; Cell Signaling Technology, Inc.), anti-YAP/TAZ antibody (cat. no. D24E4; Cell Signaling Technology, Inc), anti-Survivin antibody (cat. no. EP2880Y; Abcam), anti-CTGF antibody (cat. no. L-20; Santa Cruz Biotechnology, Inc.), and anti-CYR61/CCN1 antibody (cat. no. ab24448; Abcam). The following primary monoclonal antibodies were used for immunofluorescence: Anti-YAP antibody (cat. no. sc-101199; Santa Cruz Biotechnology, Inc.), anti-TAZ antibody (cat. no. ab84927; Abcam), and anti-CD31 antibody (cat. no. ab28364; Abcam). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (cat. no. ab6789; Abcam) and goat anti-rabbit IgG H&L (cat. no. ab97051; Abcam) were purchased for western blotting. Goat anti-mouse IgG H&L (Alexa Fluor® 488; cat. no. ab150113; Abcam) was purchased for immunofluorescence.
9
Quantitative Protein Expression Analysis
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Total protein was isolated from culture cells according to a previous study [16 (link)]. Protein samples (50 μg) were then separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, MA). Primary antibodies use anti-NIPBL antibody (1:1000, ABSea, China), anti-C-Myc antibody, anti-STAT3 antibody, anti-Mcl-1 antibody, anti-Bcl-2 antibody, anti-c-PARP antibody, anti-c-Caspase-3 antibody and anti-Survivin antibody (1:1000, Cell signaling, USA) respectively, Anti-alpha Tubulin antibody (1:1000, Abcam, UK) as the loading control. The relative level of protein expression was quantified by image analyzing software Quantity One (Version 4.5.2) (Bio-Rad, USA) and expressed as means ± SD (n = 3).
10
Ubiquitination of Survivin Protein
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Cells were harvested and sonicated in RIPA lysis buffer containing 10 mM N-ethylmaleimide (Sigma-Aldrich) and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich) on ice. Experimental methods were described in a previous study [28 (link)]. In brief, the cell lysates were incubated overnight with anti-survivin antibody (Cell Signaling Technology) and subsequently with protein PLUS-Agarose (Santa Cruz Biotechnology) for 2 h at 4 °C. Ubiquitination assay was performed using horseradish peroxidase-conjugated anti-Ub (Enzo Life Sciences) under denaturation conditions.
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