HRP PROCEDURE (Modified from J. Way's TMB)

 

  1. Sacrifice by cardiac perfusion with saline, then 1% paraformal- dehyde 1.25% glutaraldehyde in 0.1 M phosphate buffer pH 7.4, then 10% sucrose-buffer.
  2. Remove brain, block and store in 10% or 20% sucrose in phosphate buffer for 1-3 days at 4 degrees C. (refrigerator).
  3. Cut frozen sections at 25-50 um, collect in phosphate buffer.
  4. Rinse loose sections 3x in D'H2O.
  5. Pre-soak loose sections in TMB reaction media for 20 minutes on rotator.
  6. Add 2-5 ml of 0.3% H2O2, continue rotation another 20 minutes.
  7. Rinse loose sections in 0.2 M, Acetate buffer pH 3.3, 3-6x.
  8. Mount sections from the same acetate buffer onto subbed slides; let dry at room temperature overnight.
  9. Counterstain with Neutral Red (see separate procedure) or place in 2 changes of xylene 2 minutes each.
  10. Coverslip with Permount or Histoclad.


SOLUTIONS

make within 1 hour of use; twice these amounts will make enough for one tray (J. Way) or a maximum of 36 tissue sections

Stain (Reaction media):

  Na nitroferricyanide (nitroprusside)
225.0 mg

Stir
  0.2 M Acetate buffer, pH 3.3
11.25 ml
  
  D-H2O
207.8 ml
  
       
  100% EtOH
5.62 ml

heat (#3 setting for 10 min) add to Na nitro. soln
  TMB (fram freezer)
11.25 mg
  
   
 
0.3% Hydrogen peroxide:
 
  D'H2O
50.0 ml
  
  H2O2 (50% stock soln. )
0.3 ml