Five-micron formalin-fixed, paraffin-embedded tissue sections were deparaffinized, rehydrated through graded alcohol changes and then subjected to heat-induced epitope retrieval in 0.01 M citrate buffer (pH 6.0) for 30 minutes at a sub-boiling temperature. Immunohistochemistry (IHC) staining was carried with biotin-free catalyzed signal amplification kit (CSA II; DAKO, Carpinteria, CA). Slides were counterstained with hematoxylin before mounting. Appropriate positive (thyroid Graves' disease) and negative (no primary antibody) controls were included with each IHC run. NIS expression was determined using a custom affinity-purified polyclonal anti-human NIS antibody (AnaSpec, Fremont, CA) generated against the last 13 amino acids of the carboxy-terminal end of the protein.
Csa 2
Manufactured by Agilent Technologies
Sourced in United States
The CSA II is a compact and versatile analytical instrument designed for chemical analysis. It features a high-resolution optical design and advanced data processing capabilities to enable accurate and reliable measurements. The CSA II is suitable for a wide range of applications, but a detailed description of its intended use has not been provided.
Automatically generated - may contain errors
Lab products found in correlation
Dp72 1.4 mega pixel ccd, by Olympus (2 mentions)
W6b3c1, by Miltenyi Biotec (2 mentions)
Pcr blunt 2 topo vector, by Thermo Fisher Scientific (1 mentions)
Alkaline phosphatase conjugated anti dig antibody, by Roche (1 mentions)
Tsa cy5, by PerkinElmer (1 mentions)
Fluorescein isothiocyanate (fitc), by Leica (1 mentions)
Dm6000b, by Olympus (1 mentions)
Dako autostainer platform, by Agilent Technologies (1 mentions)
Rabbit anti tnfr2, by Merck Group (1 mentions)
Mouse anti neurofilament nf, by Merck Group (1 mentions)
Envision system horse radish peroxidase labelled polymer, by Agilent Technologies (1 mentions)
Mouse anti cd68 clone pg m1, by Agilent Technologies (1 mentions)
Rabbit anti gfap, by Agilent Technologies (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
5 protocols using csa 2
1
Immunohistochemical Analysis of NIS Expression
2
In situ Hybridization Technique for Coronal Sections
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For single ISH, coronal fresh-frozen sections (16 µm) were prepared. These sections were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The cDNA fragments of genes were cloned into the pCR-BluntII-TOPO vector (Invitrogen). The digoxygenin (DIG)- and fluorescein-labeled riboprobes were produced using these plasmids as templates for in vitro transcription. Fixed-sections were acetylated, and incubated in a hybridization buffer containing DIG or fluorescein-labeled riboprobes at 60°C. The sections were washed, treated with alkaline phosphatase-conjugated anti-DIG antibody (Roche) and then visualized by 4-nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as blue signals. In order to carrying out double-ISH study, the sections treated as described above were incubated with anti-FITC antibody HRP conjugate (Perkin Elmer), and treated with CSA II (DAKO) to detect fluorescein-labeled riboprobe as FITC signals (Green) or 3, 3′-diaminobenzidine (DAB) signals (Brown). After processing, the sections were mounted, and examined by light or confocal laser microscopy. NBT/BCIP signals were changed to red under imaging software as necessary.
3
Multi-marker Immunofluorescence Analysis of GBM
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Double-immunofluorescence was performed on tissue microarray containing nine GBMs. The preparations as well as the first step in the stainings are as described above. After detection of JAM-A (1 + 200) using Catalyzed Signal Amplification II kit with FITC (CSA II, Dako), sections were incubated with antibodies against nestin (196,908, 1 + 200, R&D systems, USA), CD133 (W6B3C1, 1 + 40, Miltenyi Biotec, Germany), GFAP (Z0334, 1 + 8000, Dako), SOX-2 (245,610, 1 + 400, R&D systems), or IBA-1 (019-19741, 1 + 4000, Wako Pure Chemical Industries, Japan) followed by Tyramide Amplification Signal Cyanine-5 (TSA-Cy5, Perkin Elmer, USA). Nuclei were counterstained with 4.6-diamidino-2-phenylindole (DAPI) (VWR International, USA). Fluorescence images were taken with a Leica DM6000B microscope connected to an Olympus DP72 1.4 Mega Pixel CCD (Olympus, Japan) camera using DAPI (Omega XF06, Omega Optical, USA), FITC (Leica, Germany), and Cyanine-5 (Omega XF110-2) filters. Due to cross-reaction, a different JAM-A antibody-clone (EP1042Y, 1 + 400, Abcam, United Kingdom) was used for the double staining with CD133.
4
Multimarker Immunofluorescence Staining Technique for Glioblastoma
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Double-immunofluorescence stainings were performed on a TMA containing tissue from nine GBMs, normal colon, placenta, cerebellum, and rat hippocampus as previously described [20 (link), 28 (link), 29 (link)]. The preparations as well as the first step in the stainings were as described above. After detection of TfR1 (CD71) (1+200), FTH (1+12000), or FTL (1+16000) using Catalyzed Signal Amplification II kit with FITC (CSA II, Dako), a second round of HIER followed by quenching of endogen peroxidase was performed. Sections were then incubated with antibodies against nestin (96908, 1+200, R&D systems, Minneapolis, Minnesota, USA), CD133 (W6B3C1, 1+40, Miltenyi Biotec, Teterow, Germany), GFAP (Z0334, 1+8000, Dako), or IBA-1 (019–19741 1+12000, Wako Pure Chemical Industries, Osaka, Japan). Tyramide Amplification Signal Cyanine 5 (TSA-Cy5, Perkin Elmer, Waltham, Massachusetts, USA) was used as detection system. Sections were mounted with VECTASHIELD Antifade Mounting Medium with 4.6-diamidino-2-phenylindole (DAPI, VWR International, Radnor, Pennsylvania, USA). Fluorescent images were taken with a Leica DM6000B microscope connected to an Olympus DP72 1.4 Mega Pixel CCD (Olympus, Tokyo, Japan) camera using DAPI (Omega XF06, Omega Optical, Brattleboro, Vermont, USA), FITC (Leica, Wetzlar, Germany) and Cy5 (Omega XF110-2) filters.
5
Immunohistochemical Staining of Inflammatory Markers
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Immunohistochemical staining was performed using the Dako autostainer platform (Dako, Denmark) as previously described [24 (link)]. Sections were stained using the following primary antibodies: mouse anti-CD68 (clone PG-M1, 1:100, Dako), mouse anti-CD45 (clone 2B11, 1:200, Dako), rabbit anti-Iba1 (ionized calcium binding adaptor molecule 1, 1:1000, Wako), rabbit anti-GFAP (1:2000, Dako), mouse anti-neurofilament (NF) (phosphorylated and non-phosphorylated NF-heavy chain; clone N52, 1:1000, Sigma-Aldrich), mouse anti-IL-1α (clone 4414, 1:1200, R&D Systems), mouse anti-IL-1β (clone 2E8, 1:50, BioRad), rabbit anti-TNF (1:100, ThermoFisher Scientific), rabbit anti-TNFR1 (clone H-271, 1:50, Santa Cruz), rabbit anti-TNFR2 (1:50, Sigma-Aldrich), and rat anti-IL-1Ra (clone 40,007, 1:1500, R&D Systems). The antigen-antibody complex was visualized using EnVision+System horse-radish peroxidase-labelled Polymer (Dako), PowerVision+Poly-HRP IHC (AH Diagnostics), or CSAII (Dako) detection systems.
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