Alex 594

For immunofluorescence staining, paraffin-embedded brain sections were prepared as described in our previous studies11 (link). The primary antibodies used in the experiments included anti-HMGB1 Ab (R&D Systems Inc., Minneapolis, MN), anti-AQP4 Ab (Abcam Plc, Cambridge, UK), anti-IL-1β Ab (R&D Systems Inc.), anti-microtubule-associated protein 2 (MAP2) Ab (Abcam Plc), anti-glial fibrillary acid protein (GFAP) Ab (Abcam Plc), anti-ionized calcium-binding adaptor molecule 1 (Iba1) Ab (Wako, Osaka, Japan) and anti-myeloperoxidase (MPO) Ab (Abcam Plc). In order to investigate the cellular source as well as the localization of IL-1β, double immunohistochemical staining was carried out with cell marker antibodies including MAP2, GFAP, iba1 or MPO antibodies and an antibody against IL-1β. The sections were then incubated with secondary Abs conjugated with Alexa-488, Alexa-555 or Alex594, which were purchased from Invitrogen (Tokyo, Japan). Finally, the sections were mounted using VECTASHIELD Hard Set Mounting Medium with DAPI (Vector Laboratories Inc., Burlingame, CA) and observed under an LSM 780 confocal microscopic system (Carl Zeiss Inc., Jena, Germany). The counting was performed in a blinded manner.

Immunofluorescence was performed as previously described (Hong et al., 2003 (link); Iida and Lilly, 2004 (link)). Primary antibodies used were as follows: rabbit anti-GFP (Invitrogen, 1:1000); rabbit anti-γ−H2Av (Active Motif, 1:500); rabbit anti-C(3)G 1:3000 (Hong et al., 2003 (link)); mouse anti-1B1 (Developmental Studies Hybridoma Bank, 1:100); mouse anti-γ-H2Av (Developmental Studies Hybridoma Bank, 1:5000); mouse anti-C(3)G (kindly provided by R. Scott Hawley, 1:200) (Page and Hawley, 2001 (link)). Alexa-488 and Alex-594 (Invitrogen, 1:1000) secondary antibodies were used for fluorescence. After staining, ovaries were mounted in prolong gold antifade reagent with DAPI (Life Technology). Images were acquired on either a Leica SP5 confocal microscope or Zeiss LSM 880 with Airyscan confocal microscope.