rinsed gently with buffer solution from a wash bottle (the flow was not focused directly

on the tissue) and the samples were then placed in a fresh buffer bath.

STEP 3 Peroxidase Labelled Polymer:

 Excess buffer was tapped off and the slides were wiped as in the previous steps.

Labelled polymer was applied using enough to cover the entire gingival tissue samples.

The samples were then incubated for thirty minutes and rinsed off as in step 2.

STEP 4 Substrate-Chromogen:

 Excess buffer was tapped off and the slides were wiped as in the previous steps.

Enough of the prepared substrate-chromogen was applied to cover the entire gingival

tissue samples. The slides were then incubated for ten minutes. The slides were then

rinsed gently as in the previous steps.

STEP 5 Hematoxylin Counterstain:

 The slides were immersed in a bath of aqueous hematoxylin (DAKO Code No.

S3309). The slides were then rinsed gently in a distilled water bath. The slides were

dipped ten times into a bath of 37mM ammonia as a bluing agent. The slides were then

rinsed in a bath of distilled water for five minutes.

STEP 6 Mounting:

 The gingival tissue samples were then mounted and coverslipped with an aqueous-

based mounting medium (DAKO Glycergel Mounting Medium, Code No. C0563).

 Evaluating the Slides

 Each section of the gingival tissue samples was evaluated for the presence of

intracellular brown DAB precipitate indicative of antibody binding. The staining

intensity of anti-IGF-1 was assessed using the following evaluation; weak, moderate or