Gap43

The primary cortical neurons cultured on the coverslips were fixed at defined time points with 4% PFA for 2 h at 4°C. The sections were dewaxed in xylene three times each for 5 min and re-hydrated through a series of alcohol with descending concentrations (100 to 95% to 80 to 70% and then tap water twice, each step for 5 min). The prepared tissue sections and cells were blocked in PBS with 10% normal horse serum at room temperature for 1 h and then incubated at 4°C overnight with the following primary antibodies in 10% normal horse serum: microtubule-associated protein 2 (MAP2, 1:200, Boster Biological Engineering Co.), Tuj1 (1:200, Millipore), GFAP (1:500, Boster Biological Engineering Co.), GAP43 (1:200, NOVUS), MBP (1:200, NOVUS), ionized calcium binding adaptor molecule 1 (Iba-1, 1:200, CST), CD163 (1:30, Santa Cruz), and CD68 (1:300, Boster Biological Engineering Co.). The secondary antibodies conjugated with Alexa fluor® fluorochrome (1: 300) were used to detected corresponding primary antibodies. The immunostaining results were checked under a fluorescence microscope (Olympus IX73).

The spinal cord tissues were fixed in 10% formaldehyde, embedded in paraffin and cut into 5‐μm‐thick transverse sections. Hematoxylin and eosin (H&E) staining and Nissl staining were performed according to the manufacturer's instructions. For immunofluorescence, 10‐μm‐thick transverse frozen sections were incubated with primary antibodies targeting MAP‐2 (1:200) and Nrf2 (1:200, R&D systems), and the longitudinal sections, with antibodies against GFAP (1:500, Abcam), NF‐200 (1:200, Boster), Tuj1 (1:200, CST), GAP43 (1:300, Novus), BrdU (1:200), Nestin (1:200, Novus), NeuN (1:300) and NSE (1:100, Abcam). After incubation with species‐specific secondary antibodies conjugated with Alexa Fluor 488 (1:300, CST) or Cyanine3 (1:200, Invitrogen) for 1 hour at room temperature, the sections were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and observed under a laser‐scanning confocal microscope (Leica).

Fluorescence immunohistochemistry was performed as previously described (Cheng et al, 2011 (link)). The following primary antibodies were used: hAPP, 1:1000 (6E10, Covance); OMP, 1:5000 (Wako); Gap43, 1:1000 (Novus Biologicals); cleaved caspase-3, 1:1000 (Cell Signaling Technology); Camk2a, 1:1000 (Abcam); and cleaved caspase-9, 1:200 (Cell Signaling Technology). Sections were examined using confocal microscopy (LSM510 microscope, Zeiss).

The sections were dewaxed in xylene three times each for 5 min and rehydrated through a series of ethanol of decreasing concentrations (100% to 95% to 80% to 70% ethanol and then tap water once, each step for 5 min). After the sections were heated by microwave to near boiling for 20 min in antigen retrieval buffer (10 mM citric acid, pH 6.0), all sections were cooled down to room temperature, and blocked in PBS with 10% normal horse serum at room temperature for 1 h, and then incubated with the following primary antibody at 4 °C overnight: glial fibrillary acidic protein (GFAP, 1:500; Boster Biological Engineering Co.), Nestin (1:200; Abcam) microtubule-associated protein 2 (MAP2, 1:200; Boster Biological Engineering Co.), growth-associated protein 43 (GAP43, 1:200; NOVUS), myelin basic protein (MBP, 1:200; NOVUS), ionized calcium binding adaptor molecule 1 (Iba-1, 1:200; Cell Signaling Technology), CD163 (1:30; Santa Cruz) and CD68 (1:300; Boster Biological Engineering Co.). The secondary antibodies tagged with different Alexa fluor^® fluorochrome diluted in PBS with 10% normal horse serum were used to detect the corresponding primary antibody. The immunostained sections were examined under a fluorescence microscope (Olympus IX73).