Glial fibrillary acidic protein (gfap)

For immunofluorescence (Shah et al., 2017 (link)), the rats in each group were euthanized, rinsed with warm saline via rapid left ventricular perfusion, and fixed with 4% paraformaldehyde in PBS solution for 60 min. This process was carried out at 3 days and 30 min post injury (dpi) for immunofluorescence studies. The striatal level of the brain was then dissected and fixed with 4% paraformaldehyde in PBS solution for 1 week. After that, the tissues were dehydrated with gradient ethanol, made transparent with xylene, embedded in paraffin, and cut into 0.5-μm sections using a biological tissue microtome. After dewaxing and hydration, the slides were immersed in 5% BSA for 1 h to block nonspecific antibodies. All paraffin sections were then incubated with primary antibodies overnight at 4°C, namely, GFAP (1:1000, rabbit source, Servicebio, Wuhan, China) and C3(1:400, mouse source, Santa Cruz, United States). These sections were then incubated with Alexa Fluor 594 and Alexa Fluor 488 (1:300, goat anti-mouse and goat anti-rabbit source, Abcam, United Kingdom) for 2 h.

The slices were incubated with 0.2% Triton for 10 min after rewarming, then blocked with goat serum for an hour and incubated with primary antibodies, including MPO (1:100, Abcam), NeuN (1:100, Abcam), GFAP (1:500, Servicebio), Iba1 (1:1000, Wako), MBP (1:300, Servicebio), and SMI32 (1:200; BioLegend) overnight at 4° C. After washing with PBS, a fluorescent secondary antibody was added dropwise, and the sections were incubated for two hours in the dark. The slides were rewashed, counterstained with DAPI for 10 min, washed with PBS, and mounted with an anti-fluorescence quencher.

Reagents include lentivirus plasmid (Cyagen Biosciences), potassium ferrocyanide (Sigma-Aldrich), DAPI (2 mg/ml, Servicebio), DHE (Cayman Chemical), protease inhibitors (Thermo Fisher), and phosphatase inhibitors (Servicebio). The TUNEL kit was purchased from Vazyme Biotech. The BCA protein assay kit was purchased from CoWin Biosciences. RNA extraction kit was purchased from Tiangen Biotech.
Antibodies used were as follows: hepcidin (1 : 100, Affinity), TfR1 (1 : 10000, Invitrogen), FPN1 (1 : 5000 for WB, 1 : 200 for IF, Alpha Diagnostic International), FTL (1 : 5000 for WB, 1 : 200 for IF, Abcam), GFAP (1 : 100, Servicebio), NeuN (1 : 200, Abcam), Bcl-2 (1 : 2000, ImmunoWay), Bax (1 : 1000, Servicebio), p-JNK (1 : 1000, Cell Signaling Technology), JNK (1 : 1000, Arigo Biolaboratories), NOX2 (1 : 2000, GeneTex), 4-HNE (1 : 200 for IF, 1 : 1000 for WB, Arigo Biolaboratories), BDNF (1 : 1000, Servicebio), β-actin (1 : 1000, Cwbiotech), and β-tubulin (1 : 1000, Servicebio).

Total proteins were harvested from the cortex and hippocampus. The proteins were transferred to polyvinylidene fluoride membranes (Millipore) that were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature and incubated overnight with the following primary antibodies at 4℃: anti‐p‐RIP3 (Abcam), anti‐p‐MLKL (Abcam), anti‐tumour necrosis factor‐α (TNF‐α) (Abcam), anti‐IL‐1β (Abcam), anti‐IL‐6 (Abcam) and anti‐β‐actin (Cell Signaling Technology). After incubation with secondary antibodies (Solarbio), the positive bands were visualized using an ECL substrate (Solarbio). For immunohistochemical analysis, brain tissue sections were incubated overnight with the following primary antibodies at 4℃: anti‐p‐RIP3 (Abcam) and anti‐p‐MLKL (Abcam). Subsequently, a PV9000 kit (ZSGB‐BIO) was used for follow‐up. For immunofluorescence analysis, brain tissues were blocked with goat serum for 1 hour and incubated with primary antibodies against p‐RIP3 (Abcam), p‐MLKL (Abcam), NeuN (Abcam), Iba‐1 (Wako; Servicebio) and GFAP (Servicebio) overnight at 4℃. Then, the tissues were incubated with Alexa Fluor® 594 goat anti‐rabbit IgG and Alexa Fluor® 488 goat anti‐mouse IgG secondary antibodies (Thermo Fisher Scientific). Nuclei were counterstained with DAPI (Thermo Fisher Scientific).

Freshly dissected penile and MPG tissues were harvested and fixed with 4% paraformaldehyde for further histological staining analysis. Masson trichrome staining was first carried out to determine smooth muscle and collagen expression levels in penile tissues. A 5‐μm slice was prepared and stained following the manufacturer's instructions, and the smooth muscle was stained red while the connective tissue appeared blue. For immunofluorescence staining, the penile sections were incubated with primary antibodies including α‐SMA (1:300, Servicebio, GB111364), eNOS (1:500, Abcam, ab66127), nNOS (1:500, Servicebio, GB11145), Caspase‐3 (1:200, Servicebio, GB11009‐1), and the MPG sections were covered by primary antibodies including Tuj1 (1:2500, Servicebio, GB11139), and GFAP (1:2500, Servicebio, GB11096) at 4°C overnight. After rinsing the slices with PBS, secondary antibodies were adopted for 1‐h immersion. Nuclei were stained with DAPI. Images were observed, and the interesting areas were captured under a confocal laser scanning microscope (Zeiss LSM 710) and a fluorescence microscope (NIKON, Ti2). FIJI/ImageJ software (National Institutes of Health) was used to carry out image analysis.