Rd10Mice and FA Treatment
Rd10 mice (The Jackson Laboratory) were housed in a specific pathogen-free facility in Animal Laboratories of Yantai Yuhuangding Hospital. The ARVO Statement for Use of Animals in Ophthalmic and Vision Research was adhered to for all animal studies. For treatment, rd10 animals received intragastric administration of25mg/kg, 50mg/kg and 100mg/kg FA (1270311, sigma) every day from postnatal day (P)4 to P24. Animals were sacrificed via sodium pentobarbital injection (200mg/kg; i.p.) at P25 and eyeballs were enucleated for further investigation.
Electroretinogram (ERG)Recordings
Before the ERG recordings, mice received dark adaptation overnight. Mice were anesthetized with intraperitoneal pentobarbital sodium(50mg/kg) prior to pupil dilation using 1% tropicamide. ERGs were recorded by a Ganzfeld stimulator(Roland Consult, Germany) that generated and controlled the light stimuli. Scotopic ERG were recorded with a single flash of 1.3ms duration with intensity of-1.52, -0.52, 0.48 and 1.0 log cd s/m2. A total of 5 responses per intensity were averaged together for each flash stimulus. Intraperitoneal sodium pentobarbital (200mg/kg) was then used to sacrifice mice. Amplitudes of the major ERG components (a- and b-wave) were measured (RETIsystem software) using automated and manual methods.
Hematoxylin &Eosin Staining
Eyeballs were fixed in formalin overnight prior to paraffin embedding and 3μm section preparation. Before staining, xylene was used for deparaffinization, and sections were rehydrated using an ethanol gradient prior to PBS washing. And then stained with hematoxylin and eosin. A microscope (Leica DM4000, Germany) was used to analyze retinal histology and to count outer nuclear layer nuclei.
Cell Culture and FA Treatment
BV2 murine microglial cells (Kunming Institute of Zoology) were cultured as previously described. In brief, cells were maintained in DMEM (High glucose) containing 10% FBS and penicillin/streptomycin. FA (1 mg/mL in PBS) was used to pretreat microglia for 2 h andthen stimulated with LPS (50ng/ml; L6529, Sigma). After 24 h, cells were collected for downstream analyses.
Immunofluorescence Staining
For retinal wholemounts, eyes were immersed in 4% paraformaldehyde (PFA) fixative for 30 minutes, retinal cups were separated carefully from eyeballs. Both retinal wholemounts and cell slides were stained using primary and secondary antibodies, washed extensively and retina flat-mounted. Primary antibodies: anti-iba1 (019-19471, Wako Chemicals), anti-iNOS (sc-7271, Santa Cruz). Secondary antibodies: donkey anti-rabbit Alexa Fluor 488IgG H&L, donkey anti-goat Alexa Fluor 555IgG H&L. The retinal wholemounts and cell slides were visualized via confocal microscopy (Carl Zeiss LSM710, German).
RNA Sequence
RNA was isolated from retinae using TRIzol Reagent (Invitrogen), and a Bioanalyzer 2100(Agilent) was used to gauge the quality of the resultant nucleic acid. RNA preparation, library construction, and sequenceing was conducted using a BGISEQ-500 instrument at the Beijing Genomics Institute (BGI, Shenzhen, China).
RT-PCR
Total RNA fromrd10 retina were isolated using the RNAiso Plus kit (TAKARA Bio inc, Japan), and the Reverse Transcriptase Superscript II Kit (TAKARA Bio inc, Japan) was then used to prepare cDNA following the instructions. Real time PCR was performed in 20μL reaction system, containing10μL of 2×SYBR Premix Ex Taq, 2μL of cDNA, and 10μmol/L of the primer pairs. Thermocycler settings were: 95°C for 30s; 40 cycles of 95°C for 5s, 60°C for 34s.
Western Blot
RIPA buffer (Biocolors, Shanghai, China) containing dissolved protease and phosphatase inhibitor mini tablets (Thermo Fisher Scientific, MA, USA) was used to lyse homogenized retinal tissue. Samples were then spun for 10 minutes at 10,000 rpm, after which a BCA assay was used for protein quantification. Equivalent protein amounts were utilized for western blotting. The primary antibodies were incubated overnight includeanti-STAT1, anti-pSTAT1(14994S, 7649S, CST), anti-IRF8, β-actin (ab28696, ab28696, Abcam, Cambridge, MA). After washed with PBST, the membranes were probed with HRP-linked secondary antibodies (1:2000) for 1 h at room temperature.
Statistics
Each experiment including immunostaining, qPCR and western blotting was replicated 3 times. All quantitative data was analyzed using2-tailed Student t test or one-way ANOVA by SPSS 21.0. Data are means ± standard error of the mean (SEM). P<0.05 was the significance threshold.