Mouse heads were soaked with chilled 1× phosphate‐buffered saline (PBS, 0.144 M NaCl, 2.683 KCl mM, 10.144 mM Na2HPO4, 0.735 mM KH2PO4, [P5368‐10PAK from Sigma]) and dissected using scalpel blades while placed onto a glass petri dish with a filter paper (Merck‐Millipore). The skull and meninges were removed from brain using Iris scissors (PMD120; Thermo Scientific) and tissue forceps 1:2 (PMD023445; Thermo Scientific). For brain dissection of PND0‐7 animals a magnifying loupe (Olympus KC 1,500 Ledplus; Olympus) was used. Brain areas were dissected as previously described (Spijker, 2011 ). Tissue weight was recorded before snap‐freezing in liquid nitrogen and stored in a −80°C freezer.
P5368 10pak
Manufactured by Merck Group
Sourced in United States
P5368-10PAK is a laboratory product manufactured by Merck Group. It is a pack of 10 units of the specified item. The core function of this product is to serve as laboratory equipment, without further interpretation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Pmd120, by Thermo Fisher Scientific (2 mentions)
Pmd023445, by Thermo Fisher Scientific (2 mentions)
Kc 1 500 ledplus, by Olympus (2 mentions)
Dako fluorescence mounting medium, by Agilent Technologies (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
S3014, by Merck Group (1 mentions)
Trypsin edta, by Yeasen (1 mentions)
5 protocols using p5368 10pak
1
Brain Tissue Dissection and Preservation
2
Immunohistochemical Analysis of Brain Sections
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The immunohistochemical analyses were performed as described in ref. 61 (link). In brief, brain sections were rinsed 5 times in PBS (Sigma-Aldrich P5368-10pak) for 5 min, and then blocked in 10% normal donkey serum and 0.3% Triton X-100 in PBS to minimize nonspecific binding. The sections were then incubated with the primary antibodies (Supplementary Table 4 ) in the blocking solution for 48 h at 4 °C. The sections were then rinsed in PBS and incubated with a corresponding secondary antibody for 4 h. After the last rinse, the sections were mounted, air dried overnight; and were then sealed with a cover slip in Dako Fluorescence Mounting Medium (S3023, Dako North America, Inc., Carpinteria, CA) and stored at −20 °C.
3
Apoptosis Detection in Penile Tissues
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The sections of penile tissues (5-μm thickness) were fixed in 4% paraformaldehyde/0.01 mmol l−1 phosphate-buffered saline (PBS; p5368-10PAK, Sigma) at room temperature for 30 min to 60 min and blocked with 3% H2O2. After washing, the sections were incubated with 20 μl of labeling buffer containing terminal deoxynucleotidyl transferase (TdT; 1 μl) and DIG-d-UTP (1 μl) for 2 h at 37°C. Then, the samples were stained with diaminobenzidine (DAB; SNM475, Biolab, Beijing, China), followed by counterstaining with hematoxylin. The nuclei of apoptotic cells were stained brown, and positive staining was observed under a microscope.
4
Mouse Brain Dissection Protocol
5
Intervertebral Disc Degeneration In Vitro Model
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Briefly, fresh NP tissues were kept in the solution containing 0.9% sodium chloride (S3014; Sigma-Aldrich, MO, USA), and then washed twice with phosphate-buffered saline (P5368-10PAK; Sigma-Aldrich). The samples were treated with 0.25% trypsin-EDTA (40127ES60; Yeasen Biotechnology, Shanghai, China) for 30 min and with 0.2% collagenase type II (17101015; Gibco, NY, USA) containing 0.1% FBS (10270-106; Gibco) and 1% penicillin–streptomycin mix (15140122; Gibco) for 3–4 h at 37°C. After isolation, cells were filtered through a 70 μm mesh filter (352350; BD Biosciences, NJ, USA). Primary NP cells were resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (12400-024; Gibco) containing 20% FBS and 1% penicillin–streptomycin mix and incubated at 37°C with 5% CO2. The third passaged NP cells were used in the subsequent experiments. To establish the models of IDD in vitro, 10 ng/mL IL-1β (ALX-520-001; Enzo Life Science, NY, USA) was treated with NP cells for 6, 12, 24, and 48 h.
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