Immunohistochemical Analysis of Klotho and Glial Activation in Mice

Immunohistochemistry and fluorescence staining were performed to examine Klotho protein expression in the choroid plexus and glial activation in the hippocampal CA1 subregion in mice as previously described (Kuang et al., 2014a (link); Mao et al., 2017 (link)). The selected coronal sections were incubated with a respective antibody against Klotho (1:200; Transgene, Raleigh, NC, United States), glial fibrillary acidic protein (GFAP; 1:100; Wanleibio, Shenyang, Liaoning, China), or Iba-1 (1:500; Wako, Wakayama, Japan) at 37°C for 2 h and then at 4°C overnight. The sections were then incubated with a secondary antibody conjugated with or without fluorescein isothiocyanate (Boster Biological Technology, Wuhan, Hube, China), followed by nucleus counterstaining with hematoxylin or 4,6-diamidino-2-phenylindole (DAPI; Boster). For semiquantitative analysis of the immunostaining results, the choroid plexus or hippocampal CA1 region was digitized using a 40× objective. Immunoreactivity was determined based on the integrated optical density (IOD) of Klotho-positive immunostaining in the choroid plexus or the percent area of GFAP- and Iba-1-positive immunostaining in the CA1 subfield using Image Pro Plus 6.0.