For reducing variations in immunohistochemical procedures, we mounted the lumbar spinal tissues from the different groups of rats into the same OCT block, sectioned these spinal tissues together using a cryostat at −30°C (HM550; Microm, Waldorf, Germany) and performed the following spinal immunohistofluorescence analysis, with a modified method described in Sung
et al. [
40] and our previous studies [
18,
23]. The spinal sections (10 μm) were incubated with primary antibody, anti-phosphorylated PTEN (anti-phospho-PTEN; Ser380) (1:200 dilution, cat. 9551; Cell Signaling Technology Inc., Beverly, MA, USA; polyclonal rabbit antibody), anti-PTEN (1:200 dilution, cat. 10005059; Cayman Chemical, Ann Arbor, MI, USA; polyclonal rabbit antibody), anti-phosphorylated mTOR (anti-phospho-mTOR; Ser2448) (1:200 dilution, cat. 2976; Cell Signaling Technology Inc., Beverly, MA, USA; polyclonal rabbit antibody), anti-OX-42 (CD11b, microglial marker, 1:200 dilution, cat. CBL1512; EMD Millipore, Temecula, CA, USA; monoclonal mouse antibody), anti-glial fibrillary acidic protein (GFAP) (astrocytic marker, 1:200 dilution, cat. MAB3402; EMD Millipore, Temecula, CA, USA; monoclonal mouse antibody), or anti-TNF-α (1:200 dilution, cat. ARC3012; Life Technologies Corporation, Grand Island, NY, USA) overnight at 4°C. This was then followed by Alexa Fluor 488-labeled chicken anti-mouse IgG antibody (1:400 dilution, cat. A-21200; Molecular Probes, Eugene, OR, USA; green fluorescence) or DyLight 549-conjugated donkey anti-rabbit IgG antibody (1:400 dilution, cat. 711-506-152; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA; red fluorescence) for 40 min at room temperature. For immunostaining analysis, we use a Leica DM-6000 CS fluorescence microscope (Leica Instruments Inc., Wetzlar, Germany) for examination of these stained spinal sections, and then use a SPOT Xplorer Digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) for photographing all immunofluorescence images of phospho-PTEN, PTEN, phospho-mTOR, OX-42, GFAP, and TNF-α, respectively. We measured the pixel values of the immunoreactive-positive area (using three sections per rat) by Image J software (National Institutes of Health, Bethesda, MD, USA). Spinal neurons located in the superficial laminae, laminae I to III, respond to nociceptive stimuli and directly participate in the transmission of nociception to the brain [
41], and thereby the superficial laminae play a more important role in neuropathic pain than the deep laminae. Therefore, in accordance to the method used for neuropathic rodents [
41-
44], we focused on quantifying the immunoreactivity of the targeted protein in the superficial laminae of the spinal cord. The immunofluorescence data were expressed as a percentage change compared to sham-operated or sham-operated plus i.t. vehicle group, which were considered to be 100%. For double-immunofluorescent staining of PTEN and neuronal marker, the spinal sections were incubated with a mixture of anti-PTEN (1:200 dilution) and anti-Neuronal Nuclei (NeuN) (neuronal-specific nuclear protein; neuronal marker, 1:500, Alexa Fluor 488 conjugated antibody, cat. MAB377X, EMD Millipore, Temecula, CA, USA; monoclonal mouse antibody) antibodies overnight at 4°C, and then followed by DyLight 549-conjugated anti-rabbit IgG antibody (1:400 dilution) for 40 min at room temperature. On the other hand, for double-immunofluorescent staining of PTEN and microglial marker or astrocytic marker, the spinal sections were incubated with a mixture of anti-PTEN (1:200 dilution) and OX-42 (1:200 dilution) or GFAP (1:200 dilution) antibodies overnight at 4°C, and then followed by a mixture of Alexa Fluor 488-conjugated anti-mouse IgG antibody (1:400 dilution) and DyLight 549-conjugated anti-rabbit IgG antibody (1:400 dilution) for 40 min at room temperature. For double-immunofluorescent staining of anti-TNF-α or phospho-mTOR and astrocytic marker, the spinal sections were incubated with a mixture of anti-TNF-α (1:200 dilution) or anti-phospho-mTOR (1:200 dilution) and GFAP (1:200 dilution) antibodies overnight at 4°C, and then followed by a mixture of DyLight 549-conjugated anti-rabbit IgG antibody (1:400 dilution) and Alexa Fluor 633-conjugated goat anti-mouse IgG antibody (1:400 dilution, cat. A21052; Life Technologies Corporation) for 40 min at room temperature. The double-immunostaining images were examined and acquired with Leica TCS SP5 II confocal microscope (Leica Instruments Inc., Wetzlar, Germany). We set the color of TNF-α or phospho-mTOR for 549-nm excitation line as pseudo red and set the color of GFAP for 633 nm excitation line as pseudo green. When co-localization of two proteins occurred, the merge of above two colors, pseudo red (TNF-α or phospho-mTOR) and pseudo green (GFAP), yielded yellow color.