Following a method described elsewhere [18 (link)], doublecortin (DCX) immunohistochemistry was performed. After the sections were incubated in 3% H2O2 for 30 minutes at room temperature, they were blocked by 10% normal rabbit serum in PBS with 0.2% Triton X-100 and 1% bovine serum albumin for 1 hour. The section was incubated in goat anti-DCX antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4℃ overnight. After washing 3 times, the sections were treated with a biotinylated goat secondary antibody (1:200; Vector Laboratories) for 1 hour, followed by another hour with avidin–peroxidase complex (1:100; Vector Laboratories). The sections were visualized in 50mM Tris-HCl (pH, 7.6) containing 0.03% hydrogen peroxide and 0.03% DAB for immunostaining. The slides were dehydrated by alcohol and mounted using Permount coverslips (Thermo Fisher Scientific Inc.).
Protocol detail
Doublecortin Immunohistochemistry Protocol
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Anti antibody
Antibody
Avidin
Bovine serum albumin
Dehydrated alcohol
Goat
Hydrogen peroxide
Immunohistochemistry
Peroxidase
Rabbit
Serum
Tris
Triton x 100
Vector
Corresponding Organization :
Other organizations : Korea University, Kyung Hee University, Gachon University, Gachon University Gil Medical Center
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Variable analysis
independent variables
- Incubation in 3% H2O2 for 30 minutes at room temperature
- Blocking with 10% normal rabbit serum in PBS with 0.2% Triton X-100 and 1% bovine serum albumin for 1 hour
- Incubation in goat anti-DCX antibody (1:500) at 4°C overnight
- Treatment with biotinylated goat secondary antibody (1:200) for 1 hour
- Treatment with avidin–peroxidase complex (1:100) for 1 hour
- Doublecortin (DCX) immunohistochemistry
- Sections were washed 3 times after incubation in primary and secondary antibodies
- Visualization in 50mM Tris-HCl (pH 7.6) containing 0.03% hydrogen peroxide and 0.03% DAB
- Dehydration of slides by alcohol and mounting using Permount coverslips
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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