Vwr superfrost plus slides

The eyes were enucleated and the conjunctiva of the superior pole was marked with a 6/0 silk suture. The cornea and the lens were removed and the resulting eyecups were postfixed in 4% paraformaldehyde for 1 h. The eyecups were cryoprotected by immersion first in phosphate buffered saline (PBS) containing 15% of sucrose (Sigma, Alcobendas, Madrid, Spain) for 1 day and later in PBS containing 30% sucrose for another day. The eyecups were included in Tissue-Tek® (OCT; Sakura Finetek, Torrance, CA, USA) maintaining their orientation and rapidly frozen by immersion in isopentane cooled at −70°C. Sagital 15 microns thick cryostat cross sections of the eyecups were obtained on a cryostat (Leica Jung CM3000), and placed ordered in rows in VWR® SuperFrost® Plus slides (VWR International bvba, Galdenaaksebaan, Leuven, Belgium), where they were processed for TdT-mediated dUTP nick-end labeling (TUNEL) to label apoptotic nuclei (García-Ayuso et al., 2011 (link); Montalbán-Soler et al., 2012 (link)) or for immunohistofluorescence.

The dorsal skin biopsies harvested from various regions at respective time-points from the Bcl11a^L2/L2 v/s Bcl11a^ep−/− mice were initially fixed overnight at 4 °C in 4% paraformaldehyde, followed by subsequent treatment with a graded series of alcohol and xylene before final embedding in paraffin [32 (link)]. 5 µm thick paraffin sections were cut onto VWR Superfrost Plus Slides using D554X microtome blades (C.L. Sturkey, Lebanon, PA, USA) on RM2255 microtome (Leica Microsystems, Wetzlar, Germany). Hematoxylin and Eosin (H and E) staining were performed following deparaffinization of 5 µm thick sections through a graded series of xylene and ethanol as described for histological analysis [32 (link)].

Zebra finches (n = 6) were deeply anaesthetized with ketamine (30mg/kg) and xylazine (2 mg/kg) and perfused transcardially with 0.01M PBS (phosphate buffered saline in DEPC treated Milli Q water) followed by 4% PFA (paraformaldehyde). Brains were post-fixed in 4% PFA for 24–36 hours at 4º C and kept in 30% sucrose until they were completely saturated with sucrose for cryoprotection. Ten-micron thick coronal serial sections of the brains were cut using a cryostat (Leica) and mounted on positively charged VWR superfrost Plus slides (Cat. No. 4811–703). One series was stained with Nissl and the others were used for fluorescence in-situ hybridization (FISH).