Immunofluorescence
For evaluate the effects of α-Klotho on NF-κB activation by LPS, after
purification, astrocytes were treated for 24 hours with vehicle
(control) (PBS) or 1 nM α-Klotho. Inflammatory stimulation with LPS 1
μg/ml was then performed and the cells fixed with methanol (10 min) 4
hours later and washed with PBS three times for 5 min. The fixed cells
were incubated with serum blocking (5% normal donkey serum in triton
X-100 0.01%) for one hour and incubated overnight with primary
antibodies GFAP (1:300) (3670; Cell Signaling, and RelA (p65 (1:100)
(ab7970; ABCAM, USA). The primary antibody was removed, and the plate
was washed with serum blocking three times for 10 minutes. The cells
were incubated with secondary antibody (rabbit or mouse anti donkey-
Alexa 594 or 488, Thermo Fischer Scientific,1:1000), diluted in PBS with
Triton X-100 0.01% for 2 hours, protected from the light. The
coverslips were washed five times with PBS for 5 min and incubated with
DAPI (4’-6-Diamidino-2- phenylindole; Sigma) for 1 minute in dilution
1:10.000. Slices were transferred to glass slides and analyzed in
Fluorescence microscope Nikon Eclipse 80i (Nikon, Tokyo, Japan) with a
camera system, Nikon Digital Camera DXM 1200C.