was observed with rhlIGF-1, rhIGF-2, rhIL3 sRoa, and rh TGF-j sRII. The exact

concentration of antibody required in order to neutralize the human cell surface.

 IGF-1 R mediated bioactivity is dependent on the IGF-1 concentration and on the

number and types of IGF-1 receptors present on the cell surface (a function of cell type

and culture conditions). The Neutralization Dose for this antibody is defined as that

concentration of antibody required to yield one-half maximal inhibition of the cell surface

IGF-1 R mediated IGF response on a responsive cell line, at a specific IGF concentration.

The Neutralization Dose for this lot of anti-human IGF-1 R antibody was determined to

be approximately 0.025-0.075 [tg/ml in the presence of 6 ng/ml of rhIGF-2, using the

human MCF-7 cell line.

 The staining procedure steps were followed precisely as directed in the DAKO

EnVisionTM System, HRP, Universal, and Rabbit/Mouse (DAB) as follows:

Staining Procedure

STEP 1 Peroxidase Blocking Reagent:

 Excess buffer was tapped off the gingival tissue samples. A lintless tissue was used

to carefully wipe around the gingival tissue sample to remove any remaining liquid and to

keep reagent within the prescribed area. Peroxidase Blocking Reagent was applied to

cover the gingival tissue sample. This was incubated for 5 minutes. Then the gingival

tissue sample was rinsed gently with distilled water and placed in a fresh buffer bath.

STEP 2 Primary Antibody or Negative Control Reagent:

 Excess buffer was tapped off and slides were wiped off as in step 1. Enough

primary antibody or negative control reagent were applied to cover the gingival tissue

samples. The samples were then incubated for thirty minutes. The samples were then