For immunohistochemical detection of VDR, insulin, glucagon, somatostatin, pancreatic polypeptide, TUNEL, and Ki67, pancreas were fixed for 12–24 h in formalin, embedded in paraffin, and sectioned. Sections were then incubated overnight at 4°C with the following antibodies: rat anti-mouse VDR (clone 9A7; Merck KGaA, Darmstadt, Germany), guinea pig anti-porcine insulin (Sigma Chemical, St Louis, MO), rabbit anti-human glucagon (Signet Laboratories, Dedham, MA), rabbit anti-somatostatin (Serotec, Oxford, U.K.), rabbit anti-human pancreatic polypeptide (ICN Biomedicals), and anti-Ki67 (BD Pharmingen). As secondary antibodies, peroxidase-conjugated rabbit anti-guinea pig IgG (Dako, Glostrup, Denmark), biotinylated goat anti-rabbit (Pierce, Rockford, IL), tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-guinea pig (Molecular probes, Leiden, the Netherlands), biotinyilated goat anti-rabbit (Molecular Probes), biotinylated rabbit anti-rat (Dako), and biotinylated horse anti-mouse (Vector Laboratories, Burlingame, CA) antibodies were used. Streptavidin-conjugated Alexa 488 (Molecular Probes) or streptavidin-conjugated Alexa 568 (Molecular Probes) were used as fluorochromes. Images were obtained with a Nikon Eclipse 90i microscope (Nikon, Tokyo, Japan).
Streptavidin conjugated alexa 568
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Streptavidin-conjugated Alexa 568 is a fluorescent labeling reagent. It consists of the protein streptavidin covalently linked to the Alexa Fluor 568 dye. Streptavidin has a high affinity for the biomolecule biotin, allowing it to bind to biotinylated targets.
Automatically generated - may contain errors
Lab products found in correlation
Biotinylated horse anti mouse, by Vector Laboratories (1 mentions)
Alexa 488 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Anti ki67, by BD (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
Biotinylated rabbit anti rat, by Agilent Technologies (1 mentions)
Tcs sp5 laser confocal microscopy, by Leica (1 mentions)
2 protocols using streptavidin conjugated alexa 568
1
Multimarker Immunohistochemistry of Pancreatic Tissues
2
Quantifying Adult Neurogenesis via BrdU Immunofluorescence
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For BrdU immunofluorescence, sections were denatured in 2 M HCl in TBS for 30 min, rinsed and incubated with mouse anti-BrdU (1:100, OBT-0030, Oxford Biotechnology, UK) for 2 d. Then sections were rinsed, incubated with biotinylated anti-rat (1:250; Vector) for 90 min, rinsed, and incubated for 30 min in the dark with streptavidin-conjugated Alexa 568 (1:1,000; Molecular Probes) to visualize BrdU. The analysis was performed using confocal images from coronal sections at similar rostro-caudal levels obtained with a Leica TCS SP5 laser confocal microscopy. An experimenter blinded to the treatment groups counted the BrdU+ cells in the subgranular zones and granule cell layers. The positive cells were counted on sets of every sixth section (six sections per rat) through the rostral-caudal extent of the hippocampal DG.
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