First thing to do is set up your second material. You can do this in the Sprytile panel, in the tile grid area. Make sure to run the set material to shadeless operation as well.
Next, go to the material and load the tileset in and set it to a pixel texture. Not covering it here, it's the same steps as the basics.
There are now two tilesets you can create with in your object. Duplicating this object is the easiest thing to do. However, if you start from a new mesh object, you will have to assign the materials to the new mesh. They have to be assigned in the same order as in the original object.
