After mice were perfused with ice-cold 4% PFA/PBS, eyes were dissected out and fixed in 4% PFA/PBS at 4°C overnight. The eyes were dehydrated with increasing concentrations of sucrose solution (15–30%) overnight before embedding in OCT on dry ice. Serial cross sections (12 μm) were cut with a Leica cryostat and collected on Superfrost Plus Slides. The sections were washed twice for 10 min in DEPC-treated PBS and permeabilized twice in 0.1% Tween/PBS for 10 min. After blocking at 50°C for 1 h with hybridization buffer (50% formamide, 5 × SSC, 100 μg/ml torula yeast RNA, 100 μg/ml wheat germ tRNA, 50 μg/ml heparin and 0.1% Tween in DEPC H2O), the sections were hybridized with 2 μg biotin-labeled antisense probes at 50°C overnight. The sections were washed three times at 55°C for 10 min with hybridization buffer, 0.1% Tween/PBS, and then blocked in PBS blocking buffer containing 0.1% BSA and 0.2% TritonX-100. The hybridized probes were detected by Streptavidin-AP-conjugate (Roche), and revealed by chromogenic substrate NBT/BCIP (Roche, Basel, Switzerland). Mouse CHOP and BiP probe sequences were from Allen Brain Atlas (http://mouse.brain-map.org/ ).
Streptavidin ap conjugate
Manufactured by Roche
Streptavidin-AP-conjugate is a laboratory reagent that consists of the protein streptavidin covalently linked to the enzyme alkaline phosphatase (AP). Streptavidin has a high affinity for the molecule biotin, while AP can catalyze colorimetric or chemiluminescent reactions. This conjugate is commonly used in various immunoassay and molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Superfrost plus slides, by Leica (2 mentions)
Cryostat, by Leica (2 mentions)
Nbt bcip, by Roche (2 mentions)
Cdp star, by Roche (2 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Chromatin assembly kit, by Active Motif (1 mentions)
Gata 3, by OriGene (1 mentions)
Blocking reagent, by Roche (1 mentions)
Positively charged nylon membrane, by Roche (1 mentions)
5 protocols using streptavidin ap conjugate
1
In Situ Hybridization of Mouse Eye Sections
2
Biotin-tagged Protein Detection in P. aeruginosa
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To probe the presence of multiple biotinylated proteins, the cell lysates of P. aeruginosa PAO1 were subjected to the analysis of Western blot using Streptavidin AP-conjugate (Roche) as the primary antibody (1: 1000). The resultant chemiluminescent signals were detected with CDP-Star substrate [75 (link)]. Because that the recombinant biotin-requiring enzymes is tagged with N-terminal hexa-histidine, Western blot was carried out using mouse anti-6x his primary antibody, along with the goat anti-mouse IgG secondary antibody, prior to the assay of Streptavidin blot [59 (link), 61 (link)].
3
GATA3 Binding on Compacted Nucleosomes
4
In Situ Hybridization of AKT Isoforms
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After adult 8-week old mice were perfused with ice-cold 4% PFA/PBS, eyeballs were dissected out and fixed in 4% PFA/PBS at 4°C overnight. The eyeballs were dehydrated with increasing concentrations of sucrose solution (15%-30%) overnight before embedding in OCT on dry ice. Serial cross sections (12 µm) were cut with a Leica cryostat and collected on Superfrost Plus Slides. The sections were washed twice for 10 min in DEPC-treated PBS and permeabilized twice in 0.1% Tween/PBS for 10 min. After blocking at 50°C for 1 hr with hybridization buffer (50% formamide, 5 x SSC, 100 μg/ml Torula Yeast RNA, 100 μg/ml Wheat Germ tRNA, 50 μg/ml heparin and 0.1% Tween in DEPC H2O), the sections were hybridized with 2 µg biotin-labeled antisense probes at 50°C overnight. The sections were washed three times at 55°C for 10 min with hybridization buffer, 0.1% Tween/PBS, and then blocked in PBS blocking buffer containing 0.1% BSA and 0.2% TritonX-100. The hybridized probes were detected by Streptavidin-AP-conjugate (Roche), and revealed by chromogenic substrate NBT/BCIP (Roche). Mouse AKT1, 2, 3 probe sequences were from Allen Brain Atlas (http://mouse.brain-map.org/ ).
5
Quantitative Dot Blot Analysis of DNA Targets
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For genomic DNA, 50, 100, and 200 ng were spotted onto positively charged nylon membrane (Roche). PCR products corresponding to CL25m1, CL16m1, and CL16m3/32m1 fragments were spotted in the amounts of 0.5, 1, 2, 4, and 8 ng. Hybridization was done in 250 mM phosphate buffer pH 7.2, 1 mM EDTA pH 8, 20% SDS, and 0.5% Blocking Reagent (Roche) with 50 ng of biotin-labeled probes at 65 °C with agitation overnight. Posthybridization washes were done in 20 mM Na2HPO4, 1 mM EDTA, and 1% SDS 3 × 20 min at 62 °C. Detection was carried out using streptavidin-AP-conjugate (1:5,000, Roche) followed by chemiluminescence with AP substrate CDP-Star (1:50, Roche). Dot blot intensities were compared using ImageJ with measurement of mean gray values that were inverted and normalized for background.
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