Axioplan 2 bright field microscope

Two slices per animal were analyzed representing two different levels of the dorsal hippocampus. The sections were visualized using Zeiss Axioplan 2 bright field microscope coupled to an Axiocam MRc5 digital camera (Zeiss), and representative photomicrographs of ipsilateral and contralateral DG region of the hippocampus were taken under a ×10 magnification objective. Quantitative analysis of BrdU-positive cells was performed counting the immunoreactive cells along the whole SGZ of DG in each slice under ×40 magnification objective. BrdU-positive cells were considered to be within the SGZ if they were within two cell body diameters on the border between the granular cell layer (GCL) and the hilus (see Fig. 2b) [31 (link)]. Immunoreactive cells were counted in two slices per animals, and data were plotted as the mean of BrdU-positive cells per section ± SEM.

Paraffin sections (10μ) were dewaxed in Histo-Clear (2 × 10 minutes), then rehydrated through a decreasing ethanol gradient (2 × 100%, 1 × 90/70/50/30% in ddH₂O, 2 minutes each) and washed in dH₂O for 2 × 5 minutes. For H&E staining, slides were incubated in hematoxylin (Sigma, MHSS32) for 3 minutes and then rinsed in at least 3 changes of tap water for a total of ten minutes. Next, slides were incubated in eosin (Sigma, HT110232) for 90 s, washed in ddH₂O for 2 minutes, and allowed to dry overnight. The next day, coverslips were mounted using DPX mounting media (Millipore, 100579).
Alcian blue staining was performed using an alcian blue staining kit (Vector, H-3501). Briefly, sections were incubated in alcian blue solution (pH2.5) for 30 minutes and counter stained with nuclear fast red, as per the manufacturer’s instructions. Sections were imaged using a Zeiss Axioplan2 Brightfield microscope with Zeiss Axiocam HRc color camera.

Mice were culled and eyes were enucleated and placed into Davidson's fixative for 1 h (cryosectioning) or overnight (wax embedding). For cryosectioning, eyes were removed from Davidson's fixative and placed into 10%, 15% and 20% sucrose in PBS for 15 min, 15 min and overnight, respectively. Eyes were then embedded using OCT cryopreservant and kept at −80°C until sectioning. For wax preservation, eyes were removed from Davidson's fixative and placed successively into 70%, 80%, 90% and 100% ethanol, xylene twice and then paraffin each for 45 min.
Haematoxylin and Eosin (H&E) staining was performed on 8 µm paraffin tissue sections, which were imaged on a Zeiss Axioplan 2 brightfield microscope. Anti-GFAP antibody (ab7260, Abcam) and Alexa Fluor 488 donkey anti-rabbit antibody (A21206, Invitrogen) were used on 8 µm frozen sections at 1:100 and 1:300, respectively. Slides were imaged on a Nikon A1R microscope.