Fixed SC sections were sliced using a cryostat (Leica CM3050) into 30 μm transverse sections (4 series). The contralateral side was identified with a small cut in the ventral horn. Free-floating sections were stored at −20 °C in a cryoprotectant solution of 30% (m/v) sucrose dissolved in phosphate buffer 0.1 M and 30% (v/v) ethylene glycol, until analysis.
Immunofluorescence staining for ionized calcium binding adaptor molecule 1 (Iba-1) was performed to assess microglial activation. Briefly, one series of SC sections of each animal was washed in 0.1 M PBS, treated with 1% sodium borohydride in PBS for 30 min, and further washed in PBS and in PBS with 0.3% of Triton X-100 (PBST). Then, the sections were incubated for 2 h with a blocking solution containing 0.1 M glycine and 10% normal horse serum (NHS, Gibco, Cat. No. 16050130, Auckland, New Zealand) in PBST to minimize background staining, and incubated for 2 overnights at 4 °C with a rabbit anti-Iba-1 primary antibody (1:1000; Fujifilm Wako, Cat. No. 019-19741, Neuss, Germany) diluted in PBST with 2% NHS. Sections were subsequently washed in PBST and incubated for 1 h with Alexa Fluor-594 donkey anti-rabbit secondary antibody (1:1000; Invitrogen, Cat. No. A21207, Waltham, MA, USA). Finally, after repeated washing with PBST and PBS, sections were mounted on gelatin-coated slides and coverslipped with Prolong Gold antifade mounting reagent with DAPI (Invitrogen Ltd., Cat. No. P36941). Negative controls were performed by omitting the incubation with the primary antibody.
Images of the immunofluorescence reactions were acquired with 2.5 × and 10 × objectives, using a fluorescence microscope (Axio Imager.Z1, Zeiss), through an AxioCam MRm digital camera with AxioVision 4.8.2 software (Carl Zeiss MicroImaging GmbH, Oberkochen, Germany), under the same image acquisition settings. Iba-1 fluorescent intensity was determined in images captured with a 10 × objective, using the open-source software Fiji [47 (link)] in a blinded manner. Images were first converted to 8-bit grayscale and mean grey values were automatically measured within a rectangular region of fixed dimensions (266 × 159 µM) comprising the medial two-thirds of the dorsal horn (laminae I-III) [48 (link)]. Both ipsilateral and contralateral sides were evaluated, and the average ipsilateral/contralateral ratio was calculated to normalize the data. Sections severely damaged in the region of interest were not analyzed and only animals with a minimum of 5 sections available were included in the statistical analysis.
Immunofluorescence staining for ionized calcium binding adaptor molecule 1 (Iba-1) was performed to assess microglial activation. Briefly, one series of SC sections of each animal was washed in 0.1 M PBS, treated with 1% sodium borohydride in PBS for 30 min, and further washed in PBS and in PBS with 0.3% of Triton X-100 (PBST). Then, the sections were incubated for 2 h with a blocking solution containing 0.1 M glycine and 10% normal horse serum (NHS, Gibco, Cat. No. 16050130, Auckland, New Zealand) in PBST to minimize background staining, and incubated for 2 overnights at 4 °C with a rabbit anti-Iba-1 primary antibody (1:1000; Fujifilm Wako, Cat. No. 019-19741, Neuss, Germany) diluted in PBST with 2% NHS. Sections were subsequently washed in PBST and incubated for 1 h with Alexa Fluor-594 donkey anti-rabbit secondary antibody (1:1000; Invitrogen, Cat. No. A21207, Waltham, MA, USA). Finally, after repeated washing with PBST and PBS, sections were mounted on gelatin-coated slides and coverslipped with Prolong Gold antifade mounting reagent with DAPI (Invitrogen Ltd., Cat. No. P36941). Negative controls were performed by omitting the incubation with the primary antibody.
Images of the immunofluorescence reactions were acquired with 2.5 × and 10 × objectives, using a fluorescence microscope (Axio Imager.Z1, Zeiss), through an AxioCam MRm digital camera with AxioVision 4.8.2 software (Carl Zeiss MicroImaging GmbH, Oberkochen, Germany), under the same image acquisition settings. Iba-1 fluorescent intensity was determined in images captured with a 10 × objective, using the open-source software Fiji [47 (link)] in a blinded manner. Images were first converted to 8-bit grayscale and mean grey values were automatically measured within a rectangular region of fixed dimensions (266 × 159 µM) comprising the medial two-thirds of the dorsal horn (laminae I-III) [48 (link)]. Both ipsilateral and contralateral sides were evaluated, and the average ipsilateral/contralateral ratio was calculated to normalize the data. Sections severely damaged in the region of interest were not analyzed and only animals with a minimum of 5 sections available were included in the statistical analysis.
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