Mab360

After overnight fixation, female goldfish brains were washed and embedded in Shandon Cryomatrix (Thermo Scientific), snap frozen in liquid nitrogen, and kept at −80°C. Frozen blocks were sectioned and blocked in blocking buffer (0.3% Triton PBS containing 1% bovine serum albumin (BSA) for 45 min at RT. Brain section and fixed cell slides were covered with the anti-GFAP (mouse, 1:800, Millipore, MAB360) (Forlano et al., 2001 (link)), anti-aromatase B (rabbit, 1:800, from the lab of O. Kah (Menuet et al., 2003 (link); Pellegrini et al., 2007 (link)), anti-D1R antibody (rabbit, 1:500, Acris, AP09962PU-N), or anti-TH antibody (rabbit, 1:500, Abcam, AB152) (Yamamoto et al., 2011 (link)) then incubated with donkey anti-rabbit Alexa fluor 488 (1:500, Molecular Probes) and goat anti-mouse Alexa fluor 596 (1:500, Molecular Probes) for 1 h at RT. Slides were washed and mounted with the antifading medium Vectashield with 4,6-diamino-2-phenylindole (DAPI). Images were taken by Nikon A1RsiMP confocal microscope with Nikon's Imaging Software NIS-Elements. Neuroanatomical nomenclature follows Peter and Gill (Bannerman et al., 2006 (link)).

The fixed brain slices were dehydrated with graded alcohol, embedded in paraffin wax, and coronally sectioned at 6 μM. Immunohistochemistry of sections was performed using antibodies against glial fibrillary acidic protein (GFAP, Cat.no. MAB360, Millipore) and neuronal nuclei (NeuN, Cat.no. MAB 377, Millipore) to evaluate the infiltration of inflammatory cells and viability of neurons. The positive cells were randomly counted in 5 areas per section and 3 sections of each monkey from 6 monkeys per group, and then averaged the data.

Immunoblots was performed as described previously [16 (link)]. Briefly, for Western blot analysis, samples (30 μg protein) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and were then transferred to polyvinylidene difluoride (PVDF) membranes. The primary antibodies used were as follows: mouse anti-GFAP antibody (GFAP, Millipore, MAB360, 2041104), mouse anti-APP antibody (Millipore, MAB348, 25030193), rabbit anti-CTF antibody (Millipore, AB5352, 22051154), rabbit anti-neprilysin (NEP) antibody (Millipore, AB5458, LV1423485), rabbit anti-IDE antibody (Millipore, AB9210, 2769024), mouse anti-β-actin antibody (Millipore, MAB, LV1460528) and goat anti-Iba-1 antibody (Abcam, ab5076, GR268568-3). The secondary antibodies were anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP; Jackson ImmunoResearch, 711-035-152) and anti-mouse IgG antibody conjugated with HRP (Jackson ImmunoResearch, 715-035-151). The immune complexes were detected using enhanced chemiluminescence reagents (GE Healthcare, Chicago, IL, USA). The images were obtained and quantified using a LAS-3000 Image Analyzer (Fujifilm, Tokyo, Japan).

The following antibodies were used: β-actin antibody (cw0096m, CWbio, China), FPN1 antibody (MTP11-S, ADI, USA), DMT1 (+IRE) antibody (NRAMP21-S, ADI, USA), 4-HNE antibody (HNE-11S, ADI, USA), TfR1 antibody (ab84036, Abcam, USA), L-ferritin antibody (ab109373, Abcam, USA), H-ferritin antibody (ab183781, Abcam, USA), hepcidin antibody (ab30760, Abcam, USA), Aβ_22–35 antibody (A3356, Sigma, USA), phospho-p38 (p-p38) antibody (4511S, CST, USA), p38 antibody (8690S, CST, USA), phospho-ERK (p-ERK) antibody (9102S, CST, USA), ERK antibody (4372S, CST, USA), Bcl-2 antibody (12789-1-AP, Proteintech, China), Bax antibody (50599-2-Ig, Proteintech, China), GFAP antibody (MAB360, Millipore, USA), Iba1 antibody (MABN92, Millipore, USA), NeuN (ab104224, Abcam, USA), CD31 (77699T, CST, USA), PSD-95 (ab18258, Abcam, USA), anti-rabbit IgG (RPN4301, Amersham, UK), anti-mouse IgG (RPN4201, Amersham, UK), DyLight 488 goat anti-mouse IgG (A23210, Abbkine, USA), DyLight 549 goat anti-rabbit IgG (A23320, Abbkine, USA), and DyLight 549 goat anti-mouse IgG (A23310, Abbkine, USA).

Histological examination was performed 8 h after the last injection of GSK1016790A or HC-067047 or 3 d after the onset of SE. Mice were anesthetized and then transcardially perfused with ice-cold phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. After the brains were removed, they were placed in fixative (4 °C) overnight and processed for paraffin embedding. Coronal sections (5 μm) were cut from the level of the hippocampus for toluidine blue staining. For glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule-1 (Iba-1) staining, the brains were coronally sectioned at 40 μm, and free-floating sections were incubated with primary antibodies against GFAP (Cat: MAB360, 1:1000, Millipore, Billerica, MA, USA) or Iba-1 (Cat: ab5076, 1:1000, Abcam, Cambridge, UK) overnight at 4 °C, followed by biotin-conjugated goat anti-mouse IgG (Cat: ab6788, 1:2000, Abcam, Cambridge, UK) and biotin-conjugated rabbit anti-goat IgG antibody (Cat: ab6740, 1:100, Abcam, Cambridge, UK). The surviving neurons, GFAP-positive (GAFP⁺) and Iba-1-positive (Iba-1⁺) cells were observed using a light microscope (Olympus DP70, Olympus Corporation, Tokyo, Japan) and counted as previously described^{3 (link),19 (link)}.