Doublecortin Immunohistochemistry

Christine Ann Denny, Ph.D.
02.27.08

Note: If the brains are not perfused, you will not see dendritic labeling with any of the protocols. For the best DCX staining, perfuse mice thoroughly.

Day 1

1.  Wash in 3X in 1X PBST for 10 min each at RT.

2.  Quench in 0.3% H2O2 in 1X PBS/methanol (1:1) for 15 min at RT.

3.  Wash slices 3X in 1X PBST for 10 min each at RT.

4.  Block in 10% normal donkey serum in 1X PBST for 2 hr. at RT

5.  Place in primary antibody solution in Blocking Solution O/N at 4°C

  1. Goat polycolonal anti-doublecortin (Santa Cruz, #SC 8066) 1:500 dilution
  2. 10 ml of Goat anti-doublecortin : 5 ml of PBST

Day 2

6.  Wash slices 3X in 1X PBS for 15 min at RT.

7.  Incubate in secondary antibody for 2 hr at RT.

  1. Donkey anti-goat-biotin (Jackson Immuno #705-065-003) (1:500, but since the Ab is in 50% glycerol, practical dilution is 1:250)
  2. 20 ml of donkey anti-goat-biotin: 5 ml of 1X PBS

8.  Wash 3X in 1X PBS for 15 min each at RT.

9.  Make ABC Elite Kit during the wash (30 min beforehand)

  1. 20 ul of A
  2. 20 ul of B
  3. 8 ml PBS

10.  Put in ABC Kit for 1 hr at RT and agitate.

11.  Wash 3X for in 1X PBS for 15 min each.

12.  Prepare DAB

  1. 2 drops of buffer
  2. 4 drops of DAB
  3. 2 drops of H2O2
  4. to every 5 ml of 1X PBS

13.  Incubate in DAB for approximately 9-10 min (this varies, so just watch it).

14.  Rinse 3X for 10 min each in 1X PBS.

15.  Mount slides and dry overnight

Day 3

16.  dH2O for 5 min

17.  50% EtOH for 5 min

18.  70% EtOH for 5 min

19.  95% EtOH for 5 min

20.  100% EtOH for 5 min

21.  Citrasolv for 5 min

22.  Coverslip directly out of citrasolv using a non-aqueous medium such as Permount.or DPX

23.  Let sit overnight