After anesthesia with isoflurane, the dorsal root ganglion (DRG) was separated from the L4 to L6 of mice after perfusion with 10 mL ice-cold artificial cerebrospinal fluid (aCSF: 124 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.3 mM MgSO4, 2.4 mM CaCl2, 10 mM glucose, 24 mM NaHCO3, and equilibrated with 95% O2 and 5% CO2 for 1 hour on ice). The tissues were incubated with 725 µg of collagenase type IX (lot# SLBG3258; Sigma-Aldrich) in 250 µL of Earle's balanced salt solution (Sigma-Aldrich) containing 10% FBS (as above), MEM vitamin solution (1:100, Sigma-Aldrich), penicillin-streptomycin (1:200, Life Technologies, Carlsbad, CA), and GlutaMax (1:100, Life Technologies) at 37°C for 25 minutes. Next, the DRG neurons were mechanically separated by 10 to 20 cycles of pipetting using a small diameter Pasteur pipette and filtered through a 40-μm cell strainer (BD Falcon, Franklin Lakes, NJ). The isolated neurons were placed on 12-mm diameter coverslips (Matsunami, Japan) with 40 µL of aCSF and used for experiments within 4 hours after isolation, maintaining them at 37°C in a 95% O2 and 5% CO2 humidified chamber.
12 mm diameter coverslips
Manufactured by Matsunami
Sourced in Japan
12-mm diameter coverslips are circular glass plates commonly used in microscopy applications to cover and protect specimens mounted on microscope slides. They provide a flat, transparent surface that allows for the observation and analysis of samples under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
40 μm cell strainer, by BD (1 mentions)
Mem vitamin solution, by Merck Group (1 mentions)
Collagenase type 9, by Merck Group (1 mentions)
Earle s balanced salt solution, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Duolink pla fluorescence kit, by Merck Group (1 mentions)
All in one fluorescence microscope bz x800, by Keyence (1 mentions)
Bz x analyzer software bz h4a, by Keyence (1 mentions)
Bz h4a, by Keyence (1 mentions)
24 well plate, by Corning (1 mentions)
2 protocols using 12 mm diameter coverslips
1
Isolation of Mouse Dorsal Root Ganglion Neurons
2
Visualizing p53-p65 Complexes in NSCLC
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PC9/p53EV, PC9/p53WT, and PC9/p53R248Q cells were cultured to 70% confluence on 12-mm-diameter coverslips (Matsunami, Osaka, Japan) placed in 24-well plates (Corning, Corning, NY). PC9/p53R248Q cells were treated with either DMSO as a control, 600 nM osimertinib, or TNF-α (1 ng/ml) for 24 h. Cells were fixed for 15 min with 4% paraformaldehyde in PBS, permeabilized for 10 min with 0.3% Triton X-100 in PBS, and incubated overnight at 4 °C with 1:500 dilutions of mouse antibodies to p53 (#2524, Cell Signaling Technology) and rabbit antibodies to p65 (#8242, Cell Signaling Technology) for detection of p53-p65 complexes with the use of Duolink PLA Fluorescence Kits (#DUO92002 and #DUO92004; Sigma-Aldrich, St. Louis, MO, USA). Images of PLA signals were obtained with a BZX800 all-in-one fluorescence microscope (Keyence, Osaka, Japan). Optical sectioning images of p53-p65 complexes in multiple cells acquired from top to bottom of each cell were combined into a z-projection image with the use of full-focus imaging (BZ-H4A, Keyence). The number of PLA signals per cell and percentage of the signals in the nucleus stained with DAPI were quantified in nine fields including a total of at least 50 cells for each condition with the use of BZ-X Analyzer software (BZ-H4A, Keyence).
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