Asp300s tissue processor

Paraffin-embedded sections, whole-mount GI tracts (E13.5), and myenteric plexuses (P10 or P18–P25) were used for histologic preparations. Comparable samples obtained from the duodenum, ileum, and colon were analyzed from all genotypes, using littermates as controls. The samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24 hours at room temperature. Automated dehydration of the tissues and subsequent paraffin embedding were performed using an ASP300 S tissue processor (Leica, Wetzlar, Germany). Paraffin blocks were cut to prepare 5-μm sections using a Tissue-Tek microtome (Sakura, Alphen aan den Rijn, The Netherlands). For whole-mount histology of P10 and P18–P25 tissues, the mice were perfused intracardially with PBS and 4% paraformaldehyde. The longitudinal muscle/myenteric plexus (LMMP) preparations were isolated under a dissection microscope by peeling off the outer muscle layer of the GI tract and postfixed for 15 minutes in paraformaldehyde.

Cadaveric eyes were enucleated and fixed in 4% paraformaldehyde solution. Subsequently, the anterior segment of each eye, including the lens and cornea, was removed through a circular cut through the limbus.
To generate consecutive cross sections of the left eyes of donors 1 and 2, the posterior segment was embedded in paraffin wax using a Leica ASP300S tissue processor. Cross sections that were approximately 7 μm thick were generated by sectioning the embedded eye using an RM2225 microtome. Sections were then deparaffinized using a series of xylene and ethanol dilutions. In short, tissues were immersed in xylene for 5 minutes (repeated 3 times), followed by rehydration in a graded ethanol series (100%, 100%, 90%, 90%, 70%, and 70%). Glass slides with tissues were then immersed in distilled water.
Whole mounts (all 5 right eyes) were prepared after the removal of the vitreous humor. Several incisions were made from the ora serrata toward the optic nerve head. A circular incision around the optic nerve head allowed the removal of the sclera from the chorioretinal complex. The choroid and neurosensory retina were then separated and flatmounted on glass slides. The choroidal samples were mounted with BM on the top, and the samples of the neurosensory retina were exposed with the photoreceptors on the top (Fig S1, first column, available at www.ophthalmologyscience.org).

For histological and immunohistochemical analyses, brains from scAAV9-hNDUFS4-treated and untreated animals were fixed for a few days in 10% neutral buffered formalin, and then processed on a Leica ASP300S tissue processor and infiltrated with Paraplast brand paraffin. All sections were cut on a Leica RM2245 microtome at a thickness of 4 µm. Haematoxylin and eosin staining was performed using a standard protocol. PathoGreen stain was performed by following the manufacturer’s instructions. Immunohistochemistry was performed using a Novolink Polymer Detection System.