For the analysis of Calbindin, 3 animals per group (E15 EC and SC) were used.
http://plosone.org/article/info:doi/10.1371/journal.pone.0001160#article1.body1.sec4.sec7.p2

The authors have provided the following addition to this section:

Immunostaining of enriched and control retinal sections was performed in parallel within the same experimental set. Images of RGC portions were acquired at 20x magnification using a Zeiss HR Axiocam videocamera connected to a Zeiss Axiophot microscope and digitalized by Axiovision software. To compare different specimens, the time of exposure was optimized at the start and then held constant throughout image acquisition.

Collected images were imported to the image analysis system MetaMorph and used to evaluate pixel intensity of cellular immunofluorescence. All image analyses were done blind. For IGF-I, pixel intensity was measured within the area contoured by the edges of the RGC layer, while for DCX the area was centered in the neuroblastic layer. Immunofluorescence intensity levels were calculated as the ratio between the pixel intensity and the background level, measured in the outer nuclear layer for IGF-I analysis and in the inner part of the retina for DCX analysis. Values obtained from at least 8-10 retinal fields were used to calculate the average pixel intensity value per animal.