Immunofluorescence of was performed as described previously (Gkountela et al., 2013 (link), Gkountela et al., 2015 (link)). Dilutions and catalog numbers of primary antibodies were: mouse anti-5mC (1:100, AMM99021; AVIVA Biosciences) mouse anti-H3K9me2 (1:100, ab1220; Abcam), goat-anti-VASA (R&D Systems, AF2030, 1:100), rabbit-anti-cKIT (Dako, A4502, 1:100), goat-anti-OCT4 (Santa Cruz Biotechnology, sc-8628 [concentrated], 1:100), rabbit-anti-PRDM1 (Cell Signaling Technology, 9115, 1:100), rabbit-anti-TFAP2C (Santa Cruz Biotechnology, sc-8977), goat-anti-SOX17 (Neuromics, GT15094, 1:100), and rabbit-anti-LAMININ (Abcam, ab11575, 1:100). All samples were incubated with primary antibodies overnight at 4°C. Sections were washed, incubated with FITC/TRITC-conjugated secondary antibodies (Jackson ImmunoResearch) for 30 min and mounted in ProLong Antifade Reagent with DAPI (Invitrogen). Samples were imaged on a Zeiss Axio Imager using AxioVision 4.8 software (Zeiss).
A4502
Manufactured by Agilent Technologies
Sourced in United Kingdom, Denmark
The A4502 is a laboratory instrument designed for sample analysis. It provides core functionality for spectroscopic measurements.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
S2375, by Agilent Technologies (2 mentions)
X0909, by Agilent Technologies (2 mentions)
Enhanced chemiluminescence, by Bio-Rad (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Phospo kit, by Cell Signaling Technology (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (2 mentions)
Chemidoc, by Bio-Rad (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab1220, by Abcam (1 mentions)
Axio imager, by Zeiss (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Fitc tritc conjugated secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Ab11575, by Abcam (1 mentions)
Prolong anti fade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Benchmark xt, by Roche (1 mentions)
Inform eber probe, by Roche (1 mentions)
Hhf35, by Agilent Technologies (1 mentions)
Z0311, by Agilent Technologies (1 mentions)
16 protocols using a4502
1
Immunofluorescence Staining of Stem Cell Markers
2
Immunohistochemical Liver Cancer Analysis
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We cut 5-µm-thick sections from representative paraffin blocks of liver cancer for immunohistochemistry (Supplementary Figure 6 ). The primary antibodies were hepatocyte (IR624, 1:50, Dako), GPC-3 (ZM-0146, 1:1, ZSGB-Bio), CK7 (1:1, Dako), CK19 (1:50, Dako), EpCAM (HEA125, 1:50, abcam, Cambridge, UK), and c-kit (A4502, 1:1, Dako). Immunoreactivity was evaluated according to the percentage of positive cells on sections regardless of intensity of staining, with grading form 0 to 4+ as follows: 0, no staining or equivocal reaction; 1+, 1–5% positive cells; 2+, 6–25% positive cells; 3+, 26–50% positive cells; and 4+, >50% positive cells. Tumors with a grade of 2+ were regarded as positive for antigen expression.
3
Histological Analysis of Lung Tissue
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Human lung biopsy tissue sections were stained with hematoxylin and eosin or with c-kit antibody (A4502, Dako, Carpinteria CA). Mouse tissue was fixed in 4% paraformaldehyde and specimens were embedded in paraffin and cut into 5-μm sections and stained with hematoxylin and eosin. To identify mast cells in mice and humans, sections were deparaffinized and stained with 0.5% acidified toluidine blue.
4
Detailed Immunohistochemistry Protocol
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For immunohistochemistry, the following antibodies and dilutions were used on a Ventana Benchmark XT autostainer with ultraView Universal DAB detection kits (Ventana Medical Systems): muscle specific actin (DAKO; HHF35; 1:200), calponin (DAKO; CALP; 1:300), pan-cytokeratin (Beckman Coulter; KL-10; 1:150), desmin (DAKO; D33; 1:300), CD34 (Cell Marque; QBEnd/10; 1:800), CD117 (DAKO; A4502; 1:400), S100 (DAKO; Z0311; 1:3,000), Ki67 (DAKO; MIB-1; 1:150), CD80 (Abcam; EPR1157(2); 1:300), p53 (Leica Microsystems; DO-7; 1:25), C-MYC (Abcam; Y69; 1:100), LMP1 (Diagnostic BioSystems; CS1/CS2/CS3/CS4; 1:100), LMP2A (Acris antibodies; 15F9; 1:100), EBNA2 (Merck Millipore; R3; 1:25) and the viral transcription factor ZEBRA (Santa Cruz Biotechnology; BZ1; 1:100). CD86 (BIOZOL Diagnostica; 1B3; 1:100) was stained manually. Chromogenic in situ hybridization for EBV-encoded RNA (EBER) was performed using fluorescein-labeled oligonucleotide probes (INFORM EBER Probe, Ventana). Fluorescence in situ hybridization for C-MYC (Zyto Vision) was performed as described before43 (link).
5
Immunohistochemical Profiling of Immune Cells
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Formalin-fixed paraffin-embedded samples were sectioned at 4 μm. After antigen retrieval by autoclaving in tris-ethylenediaminetetraacetic acid based buffer (S2375; Dako, Carpinteria, CA, USA), sections were incubated with 3% hydrogen peroxide and protein-free blocking solution (X0909; Dako). Sections were stained with rabbit anti-human CD117 (1:500, A4502; Dako), rabbit anti-human CD3 (1:150, ab16669; Abcam, Cambridge, UK) or mouse anti-human Forkhead box P3 (FOXP3) (1:500, ab20034; Abcam), sequentially. Sections were then incubated with EnVision+ Dual Link System-horseradish peroxidase (HRP) (K4063; Dako), EnVision rabbit-HRP (K4003; Dako) or EnVision mouse-HRP (K4001; Dako) and developed with 3,3´-Diaminobenzidine substrate using the chromogen system (K3468; Dako). Finally, specimens were counterstained with Mayer’s hematoxylin. The peak mast cells, T cells and Tregs counts were obtained by counting at least 3 representative non-overlapping HPFs using a microscope (Olympus BX51; Tokyo, Japan) at 400× magnification in a blinded manner. Samples were evaluated by JC and TO in a blind manner. In the event of discordance in histological evaluation, the specimen underwent further review until a consensus was reached on the assessment.
6
Immunohistochemical Analysis of KIT and PTEN
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Formalin-fixed, paraffin-embedded, 4-μm tumor sections were dewaxed in xylene, rehydrated with graded alcohol concentrations, and placed in an endogenous peroxide blocking buffer for 15 minutes. Sections were washed in water, antigen-retrieved, and then placed in citrate buffer. Nonreactive staining was blocked by treating the sections with 1% horse serum in Tris-buffered saline (pH 6.0) for 3 minutes. Then, anti-KIT (1:800, A4502, DAKO, Carpinteria, CA, USA) and anti-phosphatase and tensin homolog (PTEN, 1:100, ab32199; Abcam, Cambridge, MA, USA) antibodies were then applied and the antibody binding was detected using an avidin-biotin peroxidase complex (Universal Elite ABC kit, Vectastain, Burlingame, CA, USA) for 10 minutes. Diaminobenzidine tetrahydrochloride solution (Kit HK153-5K; Biogenex, San Ramon, CA, USA) was then used as a chromogen. The tumors specimens were stained with hematoxylin and eosin (H&E) to examine the basic histomorphological features.
7
Immunohistochemical Analysis of Skin and Hair
8
Immunohistochemical Analysis of DNA Repair Proteins
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Tissue biopsies were fixed in 4% paraformaldehyde (PFA) over-night, then dehydrated in 70% ethanol and paraffin embedded with successively Leica TP1020 and Leica EG1160 instruments. The 5-μm sections were cut before haematoxylin-eosine or immunohistochemistry stainings (after antigene retrival step). The following primary antibodies were used: rabbit FANCI (SIGMA HPA039972, 1:1000), rabbit FANCD2 (Abcam ab108928, 1:200), WT1 (SantaCruz SC192, 1:400), c-Kit (Dako A4502, 1:600).
9
Multimodal Immune Profiling of Skin
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Frozen skin tissues from the patients were sectioned at 5 μm and stained with primary antibodies, including mouse monoclonal [LH7.2] antibodies against C7 (ab6312; Abcam), mouse monoclonal antibodies against CD206 (321102, Biolegend), mouse monoclonal antibodies against CD68 (ab955, Abcam), and rabbit polyclonal antibodies against c-kit (A4502, Dako). Alexa Fluor 488–conjugated rabbit anti-mouse IgG and goat anti-rabbit IgG (both from Thermo Fisher Scientific) were used as secondary antibodies. Sections were stained with DAPI (Thermo Fisher Scientific). Images were captured using an LSM 780 confocal microscope (Carl Zeiss). Negative controls omitting the primary antibody were also performed (data not shown). The skin tissue sections were fixed in Karnovsky’s fixative and examined under a TEM (H-7600, Hitachi).
10
Immunofluorescence Staining of Stem Cell Markers
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