In situ hybridization was performed using a Tecan in situ hybridization instrument (Tecan, Männedorf, Switzerland) essentially as described elsewhere [10 (link)] . In brief, 6-μm thick tissue sections were pre-digested with proteinase-K (25μg/ml for 8 minutes at 37°C). In situ hybridization was performed by incubating the two double-FAM labeled NBPF LNA probes mixed or separately at 40 or 60 nM diluted in Exiqon hybridization buffer at 57°C for 1 hour. Double-FAM labeled scramble (60 nM) and miR-126 (at 60 nM) probes were used as negative and positive controls, respectively. After stringent washes in SSC buffers, the sections were incubated with alkaline phosphatase – conjugated anti-FAM (1:800, Roche, Mannheim, Germany). Slides were developed in 4-nitro-blue tetrazolium (NBT) and 5-brom-4-chloro-3’-Indolyl-phosphate (BCIP) substrate (Roche) for 60 minutes resulting in a dark-blue precipitate. Slides were counter stained with nuclear fast red (Vector Laboratories, Burlingname, CA) if not otherwise stated.
Alkaline phosphatase conjugated anti fam
Manufactured by Roche
Sourced in Germany
Alkaline phosphatase – conjugated anti-FAM is a laboratory reagent used in various assays and detection methods. It consists of an alkaline phosphatase enzyme covalently linked to an anti-FAM (fluorescein amidite) antibody. This conjugate can be utilized to detect and quantify the presence of FAM-labeled targets in samples.
Automatically generated - may contain errors
Lab products found in correlation
Nuclear fast red, by Vector Laboratories (1 mentions)
5 brom 4 chloro 3 indolyl phosphate bcip substrate, by Roche (1 mentions)
Nuclear fast red counterstain, by Vector Laboratories (1 mentions)
Nbt bcip substrate, by Roche (1 mentions)
Eukitt, by Electron Microscopy Sciences (1 mentions)
Freedom evo, by Tecan (1 mentions)
2 protocols using alkaline phosphatase conjugated anti fam
1
In Situ Hybridization of NBPF Probes
2
In Situ Hybridization of miR-21 in MF Skin
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Six μm thick tissue sections from paraffin embedded lesional MF skin biopsies were used for in situ hybridization as described elsewhere [50 (link), 73 (link)]. In brief, the slides were deparaffinized and placed in a Tecan Freedom Evo automated hybridization instrument (Tecan, Männedorf, Switzerland), which was programmed to perform the following steps: proteinase-K treatment using 15 μg/mL for 8 min at 37°C, pre-hybridization in formamide-free hybridization buffer (Exiqon, Vedbæk, Denmark) at 57°C for 15 min, in situ hybridization with double-FAM-labeled miR-21 and scramble LNA probes (both at 40nM, Exiqon) at 57°C for 60 min, stringent washes with SSC buffers at 57°C over 33 min followed by incubation of alkaline phosphatase-conjugated anti-FAM, NBT-BCIP substrate (all from Roche, Mannheim, Germany), and finally nuclear fast red counterstain (Vector Laboratories, Burlingname, CA, USA). Finally, all slides were dehydrated and the sections mounted with Eukitt (Electron Microscopy Sciences, Hatfield, PA, USA).
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