Tissue processing and immunofluorescence was performed as described by Muñoz et al. (2015 ) with minor modifications. Briefly, spinal cord cryosections were permeabilized with 100% methanol at −20°C for 10 min and then with phosphate‐buffered saline (PBS) + 0.2% Triton X‐100 (PBST) at room temperature for 10 min, blocked in PBST + 10% goat serum, incubated with primary antibodies overnight at 4°C, secondary antibodies for 2 hr at room temperature and stained with TOTO‐3 or Hoechst 33342. Antibodies used were rabbit mAb anti‐pSTAT3 (1:200, Cell Signaling Technology, D3A7), mouse mAb anti‐Sox2 (1:200, Cell Signaling Technology, L1D6A2), mouse mAb anti‐Islet1/2 (1:50; Developmental Studies Hybridoma Bank, Iowa City, IA, USA; 39.4D5), mouse mAb anti‐CD45 (1:50; Xenopus laevis Resource for Immunobiology, Rochester, NY, USA; CL21) and AlexaFluor 488 or 555 (1:500; Invitrogen, Carlsbad, CA, USA) as secondary antibodies.
Samples were photographed using an inverted fluorescence microscope (Microfluo Olympus IX71) or a confocal microscope (FV‐1000 Olympus Confocal Laser Scanning Microscope). Total cell counts for quantification analysis were determined using the ImageJ cell counter plugin.
Samples were photographed using an inverted fluorescence microscope (Microfluo Olympus IX71) or a confocal microscope (FV‐1000 Olympus Confocal Laser Scanning Microscope). Total cell counts for quantification analysis were determined using the ImageJ cell counter plugin.