Sc 7297

Tissue slides of renal cortical sections from control and experimental
mice were immersed in 100% xylene for 5 minutes four times, then placed in 100%
ethanol for 5 minutes two times, and then into 70% ethanol 5 minutes two times,
followed by 50% ethanol for 5 minutes twice. Slides were washed with
double-distilled water for 1 minute. Then slides were subjected to
1×retrieve-All antigen unmasking system buffer (catalog no. SIG-31910-50;
Covance, Dedham, MA) at 100°C for 90 minutes. Slides were kept at room
temperature for 20 to 30 minutes for cooling and washed with 1×PBS.
Slides were immersed in 0.3% Triton X-100 for 20 minutes at room temperature.
Slides were blocked with 2% bovine serum albumin for 2 hours. After blocking,
primary antibody CD44 (mouse monoclonal, sc-7297; Santa Cruz Biotechnology) and
phos-ERK (rabbit polyclonal, ab-65142, Abcam) were added overnight at
4°C. Next day, slides were washed with 0.1% Triton X-100 for 5 minutes
three times on shaker at room temperature. Secondary antibody was added with
fluorescence conjugated at 1:500 dilution for 1 hour at room temperature. Slides
were washed with 0.1% Triton X-100 for 5 minutes three times on a shaker at room
temperature and mounted for fluorescence microscopy.