Gb12096

Immunofluorescence staining was performed with primary antibodies recognizing PLK2 (1:50; Santa Cruz; sc-374643) or GFAP (1:200; Servicebio; GB12096) and shared the same procedure with immunohistochemistry staining prior to secondary antibody incubation. Sections were incubated with primary antibodies and washed three times. Thereafter, they were incubated in the dark with Alexa Fluor 488-coupled secondary antibodies for 50 min, followed by incubation in 4′,6-diamidino-2-phenylindole (DAPI) solution for 10 min. Images were collected using fluorescence microscopy (Olympus; BX53). Then optical density of GFAP was compared and PLK2 positive cell were counted by the image J software (Nie et al., 2015 (link)).

Mice were anaesthetized and transcardially perfused with 4% paraformaldehyde (PFA) in PBS. Mouse brains were dissected and post-fixed in 4% PFA in PBS, following which they were embedded in 4% agarose in PBS and sectioned to a thickness of 50 μm using a semiautomatic vibratome (Leica VT1200, Germany). The slices were selected under fluorescence microscope according to Allen Mouse Brain Atlas. Brain slices were blocked with 3% BSA in PBS with 0.3% Triton X-100 for 2 h, following which they were incubated with primary antibody (anti-oxytocin: 1:1000, AB911, Millipore; anti-vasopressin: 1:1000, PC234L, Millipore; anti-Iba1: 1:500, ab178847, Abcam; anti-GFAP: 1:500, GB12096, Servicebio) overnight at 4°C. Slices were washed three times with PBS, following which they were incubated with the secondary antibody Alexa Fluor^TM594 conjugated goat anti-rabbit immunoglobulin G (IgG) (1:1000, R37117, Invitrogen) for 1 h at 37°C. Finally, slices were counterstained with DAPI and imaged via confocal microscopy (FV1000, Olympus, Japan). To visualize neurons labeled with neurotropic viruses, brain slices were directly counterstained with DAPI and mounted on glass slides. All images were captured using an Olympus microscopy slide scanning system (BX63, Olympus, Japan) or via confocal microscopy (FV1000, Olympus, Japan). The images were processed with ImageJ.

The localization of PERK, STING, p-TBK1, and p-IRF3 were identified by immunofluorescence. Paraffin sections of brain tissue were dewaxed with xylene and dehydrated with gradient alcohol. Antigen repair was performed with EDTA (pH 8) and heated in the microwave. After cooling naturally, the sections were washed thrice with phosphate-buffered saline (PBS, pH 7.4) and blocked using 5% bovine serum albumin (BSA; #BS114, Biosharp) for 1 h at room temperature. Subsequently, slices were incubated overnight at 4°C with mouse anti-GFAP (glial fibrillary acidic protein, #GB12096,1:500, Servicebio) to identify astrocytes, mouse anti-ionized calcium-binding adaptor molecule 1 (Iba-1; #GB12105,1:500, Servicebio) to identify microglia, mouse anti-NeuN (Neuronal Nuclei, #K009907M,1:200, solarbio) and primary antibody against PERK (1:50), STING (1:100), p-TBK1(1:100), and p-IRF3 (1:200), respectively. After three washes with PBS on the next day, slices were incubated in Cy3-conjugated anti-rabbit (GB21303,1:200, Servicebio) and AlexaFluor488-conjugated anti-mouse (GB25301,1:400, Servicebio) secondary antibodies for 50 min in the dark. Finally, DAPI (G1012, Servicebio) was added to the sections for 15 min to identify the nuclei. Images were acquired on an inverted fluorescence microscope (Eclipse C1; Nikon) at 40× objective and an imaging system (DS-U3; Nikon).