Fluorescent immunohistochemistry of somatotopical glial and neuronal activation in spinal cords

At the designated time points (n = 5 animals per group at each time point), transverse frozen sections of L4 SCs (10- or 50-μm thick) from prior lymphadenectomized or sham-operated animals for the LLNs were prepared and processed for fluorescent IHC and imaging as we previously described [45, 46]. Free-floating thick sections of L4 SCs before and after mSNIs (five sections per sample) were subjected to GFAP or Iba1 single immunolabeling: mouse monoclonal anti-GFAP (1:5000, Proteintech, 60190-1-Ig; Wuhan, Hubei, China) and rabbit polyclonal anti-Iba1 (1:1000, Proteintech, 10904-1-AP) [46]. For the quantitative analysis of somatotopically determined IHC staining, the injured tibial innervation territories and the intact sural innervation territories of L4 SC-DH gray matter were determined as previously reported [52] (Fig. 1a). In brief, with the help of the stage navigator tool in Olympus cellSens Dimension software, the dorsolateral and dorsomedial edges of L4 SC-DH gray matter were first determined on the overview images. Then, along the mediolateral axis, the injured tibial innervation territories were identified as approximately the medial 45% portion and the intact sural innervation territories as the lateral one-third portion. Fluorescent Z-stack images (two fields/section) were taken using a × 20X (NA = 0.75) UPLSAPO objective across a defined area of interest (laminae I–III) in the injured tibial innervation territories and the intact sural innervation territories of ipsilateral and contralateral L4 SC-DH gray matter (Fig. 1a). Furthermore, fluorescent Z-stack images of higher magnification for representative GFAP-positive astrocytes (at least five cells per field of × 20 objectives) were taken using a × 60 (NA = 1.42) PLAPON oil-immersion objective. Fiji software was used to quantify the number of Iba1 or GFAP-positive microglia or astrocytes, percentage areas of immunoreactive signals relative to the target areas, and the mean pixel intensities (MPIs) of the positive signal of glial staining in the target areas.

Slide-mounted thin sections of L4 SCs before and 7 days after mSNIs (five sections per sample) were subjected to simultaneous NeuN/PKCγ double immunolabeling: mouse monoclonal anti-NeuN (1:500, Abcam, ab104224) and goat polyclonal anti-PKCγ (1:200, Abcam, ab71558) [45]. Images for laminae I–III of the gray matter within ipsilateral and contralateral SC-DHs were taken using a × 10 (NA = 0.40) UPLSAPO objective across the whole mediolateral axis. Fiji software was used to quantify the MPIs of the positive signal of neuronal PKCγ staining at the inner lamina II of the intact sural innervation territories in L4 SC-DH gray matter [52]. For both spinal glial and neuronal activation, representative images of almost the same anatomical localization in the matched sections were blindly selected for further processing and the assembly of the figures of this publication [46].