Sections were permeabilized with 1% saponin in PBS or 15 min in 2 N HCl in PBS for BrdU immunostaining and blocked with 10% goat or AGI-6780? donkey serum and incubated overnight at 4 C with the primary antibody. Sections were incubated in secondary antibody and coverslipped with Vectashield. Confocal images were collected sequentially on a Zeiss 710 Laser Scanning Confocal System using a 20 0. 80 Numeric Aperture 0. 55 WD ob jective lens or EC Plan Neofluar. Results were confirmed using three different biological samples. Lin 28a construct and in ovo electroporation Chicken Lin 28a was cloned from a Stage 14 embryo as previously described and the cDNA was synthetized as described in the RT PCR section using the primers described in Additional file 3, Table S3.

The PCR product was gel purified and cloned in pDrive plasmid to generate pDlin28a plasmid. Thereafter, a HindIII BamHI fragment of 895 base pairs from pDlin28a was cloned using the same restriction sites in pcDNA3. 1 to generate pCLin28a. T7 pri mer was used to confirm the integrity of the Lin 28a se quence. Electroporations were performed 1 h PR by injecting 3 ul of a mixture of pCLin28a and pIRES GFP or 3 ul of a mixture of pcDNA3. 1 and pIRES GFP as controls at the same concentration. The injections were performed using a Pico injector system PLI 100 and glass capillary needles. Thereafter, a gold plated wire electrode, used as an anode, was placed in the ventral border of the eye and a platinum and iridium electrode, used as cathode, was inserted on the top of the brain.

Three square pulses of 15 V of 50 ms length and at 950 ms inter vals were applied using an ECM 830 electroporator. The window on the shell was sealed and the embryos were returned to the incubator and col lected 72 h post electroporation and processed for immu nohistochemistry and histology. Histology and quantification Embryos used for histological analysis were fixed in Bouins fixative, embedded in paraffin, sectioned at 12 um, stained with hematoxylin and eosin, and photographed using an Olympus BX 51 microscope. To quantify the amount of transdifferentiated RPE, we analyzed the area from three histological sections of four different eyes. Images were captured using an Olympus camera and Magnafire image capture software and proc essed using ImageJ software available in.

The transdif ferentiated find more area was calculated using the free hand tool and a two tailed permutation test for comparing means using R version 3. 0. Background Several vertebrate species have the capacity to transdif ferentiate the retinal pigmented epithelium to retina. In the chick, the process of RPE transdifferentiation was first described based on histo logical observations. We previously demonstrated that after retina removal from chick eyes at embryonic day 4 to 4.