If you are observing a slide of tissue two layers thick, how can you determine which layer is superior?

Answer 1

The layer in focus when the stage is lower (or the objective is higher) is the top layer. You can watch the objective or the stage move when you coarse focus, to determine which direction turning of the focus knob does what, and then observe which direction you turn it when you fine focus from one layer to the other.

Answer 2

If you are using a stereomicroscope, you will see the depth information clearly. If you are using a monocular, focus slowly using the fine focus knob. The top layer gets focussed first and then the bottom layer gets focussed.