Computational protocol: BK channels in microglia are required for morphine-induced hyperalgesia

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Protocol publication

[…] MG6 microglial cells or the L4-L5 spinal dorsal horn cells were collected after morphine or siRNA treatment. The specimens were lysed in lysis buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, phosphatase and protease inhibitor cocktail) and mixed with Laemmli sample buffer. Proteins (30 μg) were loaded into each lane and separated by 7.5% or 12% SDS–PAGE gel. After transfer, the blots were incubated overnight at 4 °C with an anti-cPLA2 rabbit polyclonal antibody (1:1,000; Cell Signaling, #2832), anti-phospho cPLA2 rabbit polyclonal antibody (1:1,000; Cell Signaling, #2831), anti-P2X4R rabbit polyclonal antibody (1:2,000; Alomone, APR-002), anti-KCNMB3 mouse monoclonal antibody (1:2,000; NOVUS, NBP2–12916) or anti-β-actin mouse monoclonal antibody (1:5,000; Abcam, ab8226). The primary antibodies were diluted in Can Get Signal Solution 1 (Toyobo). After being washed, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:1,000; GE Healthcare) for 1 h at room temperature. The membrane-bound horseradish peroxidase-labelled antibodies were detected using SuperSignal West Femto (Life technologies) with an image analyzer (LAS-4000; Fuji Photo Film). The bands that were evaluated by apparent molecular size were quantified using the ImageJ 1.47 h software program (NIH). [...] All mice were deeply anaesthetized with sodium pentobarbital (200 mg kg−1, i.p.) and transcardially perfused with PBS, followed by 4% paraformaldehyde. Transverse spinal sections (40 μm, free floating) were incubated with an anti-KCNMB3 mouse monoclonal antibody (1:2,000) and anti-Iba1 rabbit polyclonal antibody (1:10,000; Wako, 019–19741), anti-GFAP rabbit polyclonal antibody (1:5,000; Dako, Z0334) or mature interleukin-1β (Santa Cruz, 1:1,000) for 5 days at 4 °C, followed by incubation with Cy3- or Alexa488-conjugated secondary antibodies (1:400; Jackson ImmunoResearch Laboratories). Some sections were incubated with an anti-NeuN monoclonal antibody Alexa Fluor 555 conjugate (1:2,000; Millipore, MAB377A5) for 2 days at 4 °C after treatment with the secondary antibody.For the intracellular dye labelling in fixed slices, the transverse L4 spinal sections (100 μm, free floating) were incubated with anti-Iba1 (1:10,000) in PBS overnight at 4 °C. The spinal sections were incubated with rabbit secondary antibodies conjugated with Alexa Fluor 488 (1:400; Jackson ImmunoResearch Laboratories) for 2 h. Microelectrode filled with 2% Lucifer yellow in distilled water were inserted into Iba1-positive cells under the microscope equipped with a × 40 water-immersion objective (Carl Zeiss). The dye was injected into a cell by passing a −2 nA negative current. The spinal sections were refixed in 4% PFA, and then heated in PBS for 15 min at 90 °C to remove the anti-Iba1 antibody. The sections were incubated with an anti-Lucifer yellow rabbit polyclonal antibody (1:50,000; Life Technologies, A-5750) for 5 days at 4 °C. After being washed with PBS, the sections were incubated with rabbit secondary antibodies conjugated with Cy3 (1:400; Jackson ImmunoResearch Laboratories) and mounted in Vectashield (Vector Laboratories). The microglial morphology was captured using a C2si Confocal Laser Microscope (Nikon) and was analysed with ImageJ. Total length of microglial processes was measured using the Simple Neurite Tracer software program plug-in. Semi-automated tracing of process was performed for the 3D image data. […]

Pipeline specifications

Software tools ImageJ, Simple Neurite Tracer
Application Microscopic phenotype analysis
Diseases Drug Hypersensitivity
Chemicals Morphine, Potassium, Arachidonic Acid