We mounted the rat lumbar spinal samples from multiple groups into the same OCT block and simultaneously sectioned them with a cryostat at −30 °C (HM550; Microm, Waldorf, Germany) in order to decrease variation in the immunohistochemical processes. Spinal immunohistofluorescence analyses were conducted using a modification of the method described by Sung et al. [
32] and our previous studies [
18,
31]. For double immunofluorescence staining of phosphorylated p38 (phospho-p38) and either microglia or astrocyte markers, spinal sections (10 μm) were incubated with a mixture of primary antibodies for anti-phospho-p38 (1:100, Thr180/Tyr182, cat. 4511; Cell Signaling Technology Inc., Beverly, MA, USA; monoclonal rabbit antibody) and anti-OX-42 (CD11b, microglia marker, 1:200, cat. CBL1512; EMD Millipore, Temecula, CA, USA; monoclonal mouse antibody) or anti-glial fibrillary acidic protein (GFAP; astrocyte marker, 1:200, cat. MAB3402; EMD Millipore; monoclonal mouse antibody) antibodies, overnight at 4 °C. Spinal section were then incubated with a mixture of Alexa Fluor 488-labeled chicken anti-mouse IgG antibody (1:400, cat. A-21200; Molecular Probes, Eugene, OR, USA; green fluorescence) and DyLight 549-conjugated donkey anti-rabbit IgG antibody (1:400, cat. 711-506-152; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA; red fluorescence) for 40 min at room temperature. For double immunofluorescence staining of phosphorylated ERK (phospho-ERK) and microglia or astrocyte markers, spinal sections (10 μm) were incubated with a mixture of primary antibodies, anti-phospho-ERK antibodies (1:100, Thr202/Tyr204, cat. 9101; Cell Signaling Technology Inc.; polyclonal rabbit antibody) and anti-OX-42 (1:200) or anti-GFAP (1:200) antibodies, overnight at 4 °C. Spinal sections were then incubated with a mixture of Alexa Fluor 488-labeled chicken anti-mouse IgG antibody (1:400) and DyLight 549-conjugated donkey anti-rabbit IgG antibody (1:400) for 40 min at room temperature. We used a Leica DM-6000 CS fluorescence microscope (Leica Instruments Inc., Wetzlar, Germany) to visualize the stained spinal sections, recorded images using a SPOT Xplorer Digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA), and then measured the pixel values of the immunoreactive-positive areas using Image J software (National Institutes of Health, Bethesda, MD, USA) with three sections per rat. Spinal neurons distributed over the superficial laminae (laminae I-III) respond to nociceptive stimuli, and these neurons directly contribute to the transmission of nociception [
33]. Therefore, the superficial laminae have more crucial roles in neuropathic pain compared to the deep laminae. Thus, we quantified immunoreactivity for the targeted proteins in the superficial laminae, as described by previous studies in rodents with neuropathy [
10,
33‐
35]. Immunofluorescence data is presented as a percentage change compared to the sham operation plus vehicle group, which were regarded as 100 %. Finally, for double immunofluorescence staining for neurons and phospho-p38 or anti-phospho-ERK, spinal sections were incubated with a mixture of anti-neuronal nuclei (NeuN; neuron-specific nuclear protein, 1:500, Alexa Fluor 488 conjugated antibody, cat. MAB377X, EMD Millipore, Temecula, CA, USA; monoclonal mouse antibody) and anti-phospho-p38 (1:100) or anti-phospho-ERK (1:100) antibodies overnight at 4 °C. Spinal sections were then incubated with DyLight 549-conjugated anti-rabbit IgG antibody (1:400) for 40 min at room temperature.