[Histonet] ki-67 staining

[Victor Wong]

Dear all,
 
I am working on imumunohistichemistry of ki-67 antibody (DAKO MIB-5, a monoclonal mouse anti-rat) on EDTA-decalcifed rat spine and femur.  I used DAKO's reagents such as Dual enzyme block, antigen retrieval solution pH6.0, normal goat serum, anti-mouse Envision plus kit and the DAB kit.  I washed with TBS (prepared from Invitrogen's 1M Tris-HCl pH7.5 + NaCl to 50mM Tris.HCl pH 7.4, 150mM NaCl without pH adjustment).  I used 10% NBF fixed rat liver as a control.  However, none in the liver was stained and there were non-specific staining in the samples.  Below is my protocol.
 
1.             Dewax and rehydrate sections
2.             a.   Prepare DAKO antigen retrieval solution by diluting 1:10 with distilled water;
b.      preheating the solution by microwave (boil);
c.       heat antigen retrieval solution in a copin jar with microwave;
d.      put slides to the solution and leave for 20 minutes under low to medium microwave power.
e.       Cool for 20 minutes
3.                  Wash with TBS
4.                  Quench sections in Dual Endogenous Enzyme Block for 10 minutes
5.                  Rinse with TBS
6.                  Blocking the slides with 5% goat serum for 20 mins
7.                  Incubate with 1° antibody (1:25) in 5% goat serum for 1 hours RT
8.                  wash in TBS @1min x 3
9.                  Incubate with EnVision HRP-conjugated anti-mouse antibody for 30 mins
10.              Wash in TBS @1min x 3
11.              Add DAB (20ul into 1ml DAB+substrate buffer, mix immediately) to develop
12.              Wash in water
13.              Counterstain in haemotoxylin
14.              Wash in running tap water
15.              Dehydrate, clear and mount
 
Could anyone help me in troubleshooting which steps went wrong or giving
suggestion on the staining protocol?  Any suggstion in the antigen retrieval
procedures are also welcomed.
 
Many thanks in advance.
 
Best Regards,
Victor