Immunostaining with Anti-Digoxigenin Alkaline Phosphatase

Materials
Anti-digoxigenin-AP conjugate (Roche #11093274910, 150U/200 uL, $168)
Levamisole (Sigma #T1512, 2 g, $10)  (blocks endogenous phosphatase activity)
BCIP (Roche #11383221001, 3 mL, $49)
NBT (Roche #11383213001, 3 mL, $48)
Atlternate BCIP/NBT (Roche, #11697471001, 20 Tabs, makes 10 mL/Tab, $67)
Blocking Reagent (Roche #11096176001, 50 g, $104, or 10x Block Buffer #11585762001, 1 set, $147)
Alternate blocking reagents:   Nonfat dry milk, mouse serum, goat serum.
Tris-HCl, NaCl, MgCl2

Solutions
Buffer 1: 100 mM Tris-HCl pH 7.6, 150 mM NaCl, store at room temp.
Blocking Buffer:  10% w/v Blocking Reagent in Buffer 1.
Alternate Blocking Buffer:   1% w/v nonfat dry milk, 1% mouse serum, 1% goat serum, 1% BSA, in Buffer 1.
Buffer 2: 100  mM Tris-HCl pH 9.5, 50 mM MgCl2 , 100 mM NaCl (use pH 10 for blue color)
Buffer 3: Buffer 2 with 0.5 mg/mL levamisole (make fresh)
Color Detection Solution: 0.45 uL/mL NBT, 3.5 uL/mL BCIP, in Buffer 3.  (Make fresh)
Alternate Color Detection Solution: 1 Tablet of BCIP/NBT, 10 mL Buffer 3.  (Make fresh)
TE:  10 mM Tris base pH=8.0, 1 mM EDTA.  Store at room temp.

Procedure

1. Cut frozen sections.  Fix in -20ºC acetone briefly and air dry.  Altertatively cut paraffin sections and deparaffinize with xylene and graded EtOH into PBS.
2. Block slides with 1/20x serum/PBS (use the appropriate blocking serum, i.e. same as 1º Ab), 10'.  Wash in PBS 2' x3.
3. Add digoxigenin labeled 1º antiobody.  Ususally 1:1000 (v/v) in PBS.  Wash in PBS 2' x3.
4. Equilibrate slides in Buffer 1, RT x 5'.
5. Incubate slides in Blocking Buffer, RT x 30'
6. Incubate  with anti-Dig-AP conjugate  (1:2000 in Blocking buffer.), 2 hrs. RT, or o.n. at 4ºC.
7. Wash in Buffer 1, 10' (x3).
8. Inactivate endogenous peroxidase by incubating in Buffer3 for ≥ 5'.
   Incubate in Color Detection Solution for 20 - 60', until desired intensity is achieved.  Protect from light. 
   Stop color development with TE pH=8.0.  
9. Post-fix with 4% paraformaldehyde for 20'. 
10. Wash 3x in PBS.
11. Dehydrate with graded EtOH and xylene and then coverslip with Permount.