Cryostat sections, 15��m thick, were cut in the frontal plane. Sections through different retinal areas were thaw-mounted on SuperFrost Plus slides (Menzel-Glaser, Germany), air-dried, and stored at ?80��C.2.2. normally ImmunohistochemistryWorking solutions and sources of primary and secondary antibodies used in the present study are summarized in Table 2. The anti-Isl1 monoclonal antibody [38] was developed by Dr. Jessell (Columbia University). It has recently been used and tested in mouse, chicken, turtle, and Xenopus, with comparable results [12, 18, 31, 33, 34, 39] (see also data sheet DSHB), and the staining pattern colocalized with the mRNA distribution [40]. The rest of the primary antibodies used in this report have been widely used in neuroanatomical studies in the central nervous system of different groups of vertebrates.
They cross react with antigens present in the X. laevis tissue, showing similar staining patterns to those previously described in other vertebrate retinas. Single- and double-immunohistochemical studies were performed as described in Bejarano-Escobar, Blasco [9] and simultaneously labeled with the nuclear dye 4��,6-diamino-2-phenylindole (DAPI). Sections were coverslipped with Mowiol for observation. Controls for the Isl1 immunostaining included (i) Western blot for the Isl1 antibody which showed a band in Xenopus, Rana, and rat that corresponded to the Isl1 LIM homeobox-1 because it coincides with the expected molecular weight (about 39kDa); [41]; (ii) incubation of some selected sections with preimmune mouse or rabbit sera (1:1000 dilution) instead of the primary antibody; (iii) controls in which either the primary or the secondary antibody was omitted; and (iv) preadsorption of the primary antibody with synthetic peptide (Isl1 peptide, Abcam, Cambridge, MA, USA) [42].
In all the latter controls, the immunostaining was omitted. Controls for the other antibodies used in combination with Isl1 can be found elsewhere: CB, CR [43], CERN-922 [9�C11], and SV2 [10, 11].Table 2Immunoreagents, working dilutions, and sources of antibodies used in the present study.2.3. Image Acquisition and ProcessingDigital images of X. laevis specimens were captured with a Digital Camera DS-5Mc (Nikon) attached to a Stereoscopic Microscope SMZ-1000 (Nikon).
Immunolabeled sections were observed using an epifluorescence, bright field Nikon Eclipse 80i microscope, and photographed using an ultra-high-definition Nikon digital camera DXM1200F. Batimastat Some of the double-immunolabelings were photographed with a Nikon D-Eclipse C1 confocal laser-scanning microscope. Graphical enhancement and preparation for publication were performed in Adobe Photoshop (v.CS4).3. ResultsAll the retinal cell types during development of Xenopus laevis are generated within 36 hours (between St24 and St40) after the birth of the first retinal ganglion cells [44].