Here is my protocol
REAGENTS:
	0.3%[vol/vol] Hydrogen  Peroxide - made fresh from 30% stock
solution with PBS as the diluent
	3.0% BSA diluted in TBS with 0.1% Triton-X

	Primary Antibody:Cleaved Caspase-3 Antibody
		New England Biotech
			
		Diluted 1:100 in 3%BSA [TBS with 0.1% Triton-X]	
	
	Secondary antibody: goat anti-rabbit IgG[H+L] Horse Radish
Peroxidase Conjugate
	Source:  Biorad Laboratories
		1:250 in  3%BSA [TBS with 0.1% Triton-X]
		
	AEC freshly made as follows:
		Add 5ml of pH 5.0 NaAcetate to 200ml of H2Odist
		Add 10ml of AEC 4mg/ml
		Filter
		Add 100ul of 6% H2O2 just prior to use

PROTOCOL:
	
	-Paraffin sections cut 4um sections deparaffinize and hydrate
through grade alcohols to TBS with 0.1% Triton-X100 [TBS-TX]

	HIER per SOP

1.Immerse slides in 3%BSA in
TBS-TX......................................................................
....	1 hour RT
2.Add 200ul of diluted caspase
-3..........................................................................
.........	 0vernight 4C
3. Rinse in
TBS-TX......................................................................
..................................	20 minutes
			2 washes 10 minutes each
4. Immerse in 0.3% H2O2in TBS-TX to block endogenous
peroxidase............................	 15 minutes
5. Rinse in
TBS-TX......................................................................
..................................	20 minutes
			2 washes 10 minutes each
6. Add 200ul diluted aRb
IgG-HRP.....................................................................
............	1hour RT
7. Develop with AEC freshly
made........................................................................
...........	10 minutes

Cynthia Favara
Rocky Mountain Laboratories
903 S. 4th Street
Hamilton, MT 59840
406-363-9317
FAX 406-363-9286

-----Original Message-----
From: r-meyer2@northwestern.edu [mailto:r-meyer2@northwestern.edu]
Sent: Friday, September 21, 2001 7:38 AM
To: histonet@pathology.swmed.edu
Subject: caspase 3


Has anyone done IHC with caspase 3.  If so, where was your antibody from and

what protocol did you use.  Thanks 

Bob Meyer,HTL 
Northwestern University