In situ hybridization was performed as described: following fixation with 4% PFA, acetylation with acetylation buffer (1.3% triethanolamine, 0.25% acetic anhydride, 20 mM HCl), treatment with proteinase K (5 μg/ml, IBI Scientific) and pre-hybridization (1 × SSC, 50% formamide, 0.1 mg/ml Salmon Sperm DNA Solution, 1 × Denhart, 5 mM EDTA, pH 7.5), brain sections were hybridized with DIG-labeled LNA probes at Tm -22°C overnight. After washing with pre-cooled wash buffer (1 × SSC, 50% formamide, 0.1% Tween-20) and 1 × MABT, sections were blocked with blocking buffer (1 × MABT, 2% blocking solution, 20% heat-inactived sheep serum) and incubated with anti-DIG antibody (1:1, 500, Roche) at 4°C overnight. Brain sections were washed with 1 × MABT and Staining buffer (0.1 M NaCl, 50 mM MgCl2, 0.1 M Tris-HCl, pH 9.5), stained with BM purple (Roche) at room temperature until ideal intensity was reached. The antisense RNA probe (Sfrp1, Wnt7a, Wnt7b, Pax6, Ngn2, and Hes5) was labeled using the DIG RNA labeling Kit (Roche, Switzerland) following the manufacturer’s instructions.
Proteinase k
Manufactured by IBI Scientific
Sourced in United States
Proteinase K is an enzyme that cleaves peptide bonds in proteins. It is commonly used in molecular biology and biochemistry applications to degrade and digest proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bm purple, by Roche (2 mentions)
Anti dig antibody, by Roche (2 mentions)
Mini beadbeater 8, by Biospec (2 mentions)
Dig rna labeling kit, by Roche (1 mentions)
Microtome, by Leica (1 mentions)
Bz x analyzer software, by Keyence (1 mentions)
Thioflavin s, by Merck Group (1 mentions)
Tissue lysis buffer atl, by Qiagen (1 mentions)
Powersoil dna isolation kit, by Qiagen (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
6 protocols using proteinase k
1
In situ Hybridization for Neural Gene Expression
2
Immunohistochemical Analysis of Brain Sections
3
Cdx2 Polymorphism Analysis in BC Cells
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The ER-positive and ER-negative BC cell lines were used to assess the Cdx2 polymorphism in the vdr gene using pyrosequencing technology as described below. For DNA extraction, cells were lysed in specific buffer (NaCl 300Mm, EDTA 25Mm, Tris 50Mm PH = 8 2% SDS and Proteinase K (IBI Scientific, Peosta, IA) and total DNA was extracted using the kit Phase Lock Gel (PLG-Prime, Gaithersburg, MD) following the manufacturer’s instructions.
4
Microbial DNA Extraction from Milk
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DNA was extracted from each collected sample separately. Also, 10 ml of milk was centrifuged at 4°C and 9,000 rpm for 30 min. The fat and majority of supernatant were removed by suction and 300 μl supernatant retained to resuspend the pellet. The milk pellet and the remaining supernatant were vortexed and transferred to a sterile micro centrifuge tube using a sterile transfer pipette, before being incubated at 40°C for 12 h with 180 μl of tissue lysis buffer ATL (Qiagen, Valencia, CA, USA), 40 μl of proteinase K (IBI Scientific), and 20 μl of lysozyme solution (10 mg/ml) to maximize bacterial DNA extraction.
Isolation of genomic DNA was performed on 250 μl of post-incubation mixture pipetted into PowerBead Tubes (PowerSoil® DNA Isolation kit, MO BIO Laboratories, Inc., Carlsbad, CA, USA) and settled in a Mini-Beadbeater-8 (Biospec Products, Battersville, OK, USA) for microbial cell disruption. DNA extraction was performed using a PowerSoil DNA Isolation Kit (MO BIO Laboratory Inc.) following the manufacturer’s recommendation. DNA concentration and purity were evaluated by optical density using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE, USA) at wavelengths of 230, 260, and 280 nm.
Isolation of genomic DNA was performed on 250 μl of post-incubation mixture pipetted into PowerBead Tubes (PowerSoil® DNA Isolation kit, MO BIO Laboratories, Inc., Carlsbad, CA, USA) and settled in a Mini-Beadbeater-8 (Biospec Products, Battersville, OK, USA) for microbial cell disruption. DNA extraction was performed using a PowerSoil DNA Isolation Kit (MO BIO Laboratory Inc.) following the manufacturer’s recommendation. DNA concentration and purity were evaluated by optical density using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE, USA) at wavelengths of 230, 260, and 280 nm.
5
In situ Hybridization with LNA Probes
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In situ hybridization was performed on frozen sections using locked nucleic acid (LNA) probes(Obernosterer et al,2007 ). After postfixation with 4% paraformaldehyde (PFA) for 10 min and acetylation with acetylation buffer for 10 min (1.33% triethanolamine, 0.25% acetic anhydride, 20 mM HCl), samples were treated with proteinase K for 5 min (10 mg/ml, IBI Scientific) and pre‐hybridized (1× SSC, 50% formamide, 0.1 mg/ml salmon sperm DNA solution, 1× Denhart, 5 mM EDTA, pH 7.5) for 6 h at room temperature. Brain sections were hybridized with DIG‐labeled LNA probes at RNA melting temperature (Tm) −30°C overnight (1:300 in hybridization buffer). The first wash was made at hybridization temperature for 15 min, after which 2 more subsequent washes were made at 4°C (1× SSC, 50% formamide, 0.1% Tween‐20). After washing 2 times with pre‐cooled 1× MABT, sections were blocked in blocking buffer (1× MABT, 2% blocking solution, 20% heat‐inactivated sheep serum) for 2 h at RT and incubated with anti‐DIG antibody (1:1,500, Roche) at 4°C overnight. Sections were washed 5 times for 20 min at RT with 1× MABT and 2 times for 10 min at RT with staining buffer (0.1 M NaCl, 50 mM MgCl2, 0.1 M Tris–HCl, pH 9.5). Finally, sections were stained with 500 μl of BM purple, which was replaced every 6 h (Roche) at room temperature until ideal intensity was reached.
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In situ hybridization was performed on frozen sections using locked nucleic acid (LNA) probes(Obernosterer et al,
6
Bacterial DNA Extraction from Skin Samples
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HS, ADD and IDD samples were washed 3 times with nuclease-free water and incubated at 56°C for 12 h with 40 μl of proteinase K (IBI Scientific), 180 μl of tissue lysis buffer and 40 μl of lysozyme (QIAamp DNA Minikit, Qiagen, Valencia, CA, USA) to maximize bacterial DNA extraction. 250 mg of post-incubation healthy skin samples and DD lesions were placed in PowerBead Tubes (PowerSoil DNA Isolation kit, MO BIO Laboratories, Inc., Carlsbad, CA, USA) and settled in a Mini-Beadbeater-8 (Biospec Products, Battersville, OK, USA) for microbial cell disruption. DNA extraction was performed using a PowerSoil DNA Isolation Kit (MO BIO Laboratory Inc.) following the manufacturer’s recommendation. DNA concentration was evaluated using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies Corporation, Carlsbad, CA, USA).
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