The Stereo Investigator user guide is within software help menu.
● You should aim to count roughly 200 cells to get a CE of <0.1 (even better if <0.05; but
much less than 0.05 means that you’re probably doing more work than you need to).
● You should use about 10 sections covering the entire structure of interest (therefore, you
are looking to count about 20 cells per section). For each brain, the choice of the first
section should be random with respect to the structure of interest; thereafter the section
interval should be constant (ie. if taking every third section from serial sections, the
choice of whether the first section is section 1, 2, or 3 should be random, but then you
would take sections 1, 4, 7…, or 2, 5, 8…, or 3, 6, 9… respectively).
● Counting frame size should give an average of 3–4 cells counted per frame. This may
appear to suggest that a grid size that gives 5–7 counting frames per section would be
ideal; but in practice more counting frames per section are needed (~10), this is because
many counting frames will fall at the edge of the structure and will have few or no cells to
count. The CE calculation is complex, but more smaller counting frames tends to give a
lower CE than fewer larger counting frames.
● The image acquisition workflow will only image one field of view per counting frame. This
means that counting frame size is limited by the field of view. At 100×, 50 × 50 μm is
about as large as the counting frame can be. At 40×, sizes up to 100 × 100 μm are OK.
If the counting frame is too large imaging will proceed as normal, but you may get odd
behaviour in the optical fractionator workflow.
● The guard zone should take you beyond fluctuations in the slice surface. Ideally it would
be at least one cell diameter (if counting cells), but for immunostained tissue a guard
zone this thick is unlikely to ever be practical.
● The probe thickness would ideally be much greater than one cell diameter (if counting
cells), but again for immunostained tissue this is unlikely to ever be practical.
● For immunostained tissue, top guard zone + probe thickness should be less than
antibody penetration depth. Obviously this has to take precedence over the previous two
rules (you can’t count cells that you can’t see).