*2.10. Immunofluorescence*

For immunofluorescence (IF) staining, sections were rehydrated and incubated for 30 min at 90 ◦C (water bath) in 10 mM Na citrate/0.05% Tween 20, pH 6.0, for antigen retrieval. Standard procedure included blocking for 30 min at room temperature (RT) with 5% normal goa<sup>t</sup> serum/0.05% Tween 20 in Tris-buffered saline (TBS-T), incubation with primary antibodies for 12 h at 4 ◦C in a humidified chamber, and 1 h of incubation with fluorescence-labeled secondary antibodies at RT. Nuclei were stained with 10 μg/mL Hoechst-33342 for 15 min at RT. Sections were cover slipped with ImmuMount (Thermoscientific). The following primary antibodies were used in double staining experiments: Polyclonal rabbit anti-GFAP (Sigma G9269; 1/500), polyclonal guinea pig anti-Iba1 (1/500; Synaptic systems 234004, Göttingen, Germany), monoclonal mouse anti CD68 (1/250; Serotec MCA341R1, Alcobendas, Spain). Secondary antibodies were labeled with fluorescent dyes: Goat anti-guinea pig IgG, Alexa-488 (Invitrogen A11073; 1/500), goa<sup>t</sup> anti-rabbit

IgG, TRITC (1/500; Sigma T5268, Madrid, Spain), goa<sup>t</sup> anti-mouse IgG, Alexa-594 (Invitrogen A11005; 1/500), goa<sup>t</sup> anti-mouse IgG, Alexa-488 (1/500; Jackson 115-545003, Cambridge, UK).

### *2.11. Microscopy and Image Analysis*

Immunohistochemical staining was evaluated using a Leica epifluorescence microscope (20<sup>×</sup>, 40× objective). Exposure conditions were kept constant for quantitative evaluation with GFAP, CD68, and Iba-1. Photographs were analyzed using Fuji Image-J, applying the same brightness/contrast adjustments and threshold values for each marker.

The intensity of immunoreactivity was measured as *integrated density* in regions of interest (ROI) in the ventral white matter at 8 mm distances anterior and posterior (ROI 0.3 mm2) and within the lesion center (ROI 0.075 mm2). Following background subtraction, signal intensities were normalized to values found in spinal cord sections from shamoperated rats (4 dpo) or rats without SCI (6 W survival). The number of CD68 positive cells was counted in the same regions. For the evaluation of apoptosis, we counted cell nuclei that were TUNEL positive and expressed the data as percentages of all nuclei in ROI of 1 mm2, which were located in ventral white matter at 4 mm anterior and posterior of the lesion and in the lesion center.

A Sholl analysis was performed to quantify morphological changes of microglia cells. Iba-1-stained sections from spinal cord gray matter were photographed at an 8 mm distance from the site of injury (laminectomy or SCI +/ − treatment) and visualized with Image-J. From three rats per treatment group, 15 cells were randomly selected and eight concentric rings superimposed over the cell nuclei (Fiji plugin *concentric circles*). The number of intersections of cell processes with each ring was counted manually, excluding extensions from neighboring cells.

### *2.12. Statistical Analysis*

Unless stated otherwise in the figure legends, data are presented as mean values ± standard error of the mean (SEM). In box and whiskers plot data are shown with median, first and third quartiles and complete range. Statistical analysis, performed with GraphPad Prism software, consisted of one-factor or two-factor ANOVA, followed by post-hoc Dunnett's or Sidak's multiple comparison tests. In graphical data representation, statistical significance is indicated as follows: \*, #: *p* < 0.05, \*\*, ##: *p* < 0.01 and \*\*\*, ###: *p* < 0.001. Normal distribution of data within groups was assessed with a Kolmogorov-Smirnov test. Performance differences of treatment groups in von Frey and Rotarod tests were assessed using confidence intervals of proportions based on binomial calculation.
