Superfrost plus microscope slide

For in situ hybridization and immunohistochemistry experiments, 2-month-old adult animals were deeply anesthetized with ketamine-xylazine (10 mg/ml and 1 mg/ml, respectively) and were transcardially perfused with PBS, pH 7.4, followed by 4% (w/v) formaldehyde in PBS, pH 7.4. Embryonic, postnatal, and adult brains were removed, immersion-fixed in fixative 4% (w/v) formaldehyde in 100 mm phosphate buffer, pH 7.4, overnight at 4°C, and subsequently cryoprotected in 30% (w/v) sucrose-PBS. Tissue samples were embedded in optimum cutting temperature compound (VWR International) and sectioned on a cryostat (CM3050S; Leica) as 20 μm sections on Superfrost Plus microscope slides (25 × 75 × 1.0 mm; Thermo Scientific) for embryonic brains and 35–50 μm free-floating sections for postnatal and adult brains.
For RNAscope experiments, adult animals were deeply anesthetized with ketamine-xylazine (10 mg/ml and 1 mg/ml, respectively) and were transcardially perfused with 10% neutral buffered formalin (Sigma-Aldrich). Brains were then removed and immersion-fixed in 10% neutral buffered formalin overnight at room temperature and then transferred into 70% ethanol for storage. Brains were subsequently embedded in paraffin wax and processed into 4 μm sections collected onto Superfrost Plus microscope slides (25 × 75 × 1.0 mm; Thermo Scientific) using the Leica RM2255 microtome.

Animals were deeply anesthetized by intraperitoneal injection of pentobarbital (150 mg/kg bodyweight) and subsequently transcardially perfused with isotonic Ringer solution (5 ml/l Heparin, B. Braun) and 4% Paraformaldehyde (PFA, in 0.2 M phosphate buffer, pH 7.4). Brains were removed from the skulls and post-fixed for 4 h at 4°C. For cryoprotection, brains were transferred to 30% sucrose and kept overnight at 4°C. Coronal sections with a thickness of 40 μm were cut using a sliding microtome (Microm HM430, Leica). Sections were collected and stored as free-floating sections in cryoprotectant solution at −20°C until further processing. For immunohistochemical staining brain sections were washed with 0.1 M phosphate buffer (PB) and then incubated with a blocking and permeabilization solution (TNB, 0.1% TBST, 3% normal goat serum) for 30 min at RT shaking. Sections were incubated with primary antibody (rat-CD31, BD Biosciences, 1:100) overnight at 4°C. The next day sections were washed and incubated with corresponding secondary antibodies for 2 h at RT. All antibodies were diluted in blocking and permeabilization solution (TNB, 0.1% TBST, 3% normal goat serum). Nuclei were counterstained with DAPI (1:2000 in 0.1 M PB). Sections were mounted in 0.1 M PB on Superfrost PlusTM microscope slides (Thermo Fisher) and coverslipped using Mowiol.

Animals were deeply anaesthetized by intraperitoneal injection of pentobarbital (150 mg/kg bodyweight) and eyes enucleated using curved forceps. For retinal flatmount preparation, eyes were prefixed with 4% PFA for 20 min at 4 °C following excision of the cornea, lens, sclera and vitreous and isolation of the retina. Retinas were flatmounted on permeable cell culture inserts (BD Falcon) and sequentially fixed with 4% PFA from both sides for 20 min at 4 °C, respectively. For immunohistochemical staining retinal flatmounts were washed with 0.1 M phosphate buffer (PB) and then incubated with a blocking and permeabilization solution for 30 min at RT shaking. Tissue was incubated with primary antibodies against CD31 (rat, 1:100, BD Biosciences) and Isolectin B4 (1:100, Vector Labs), overnight at 4 °C. The next day the tissue was washed and incubated with corresponding secondary antibodies conjugated to Cy3 (Thermo Fischer) for 2 h at RT. All antibodies were diluted in blocking and permeabilization solution (TNB, 0.1% TBST, 3% normal goat serum). Flatmounts were mounted in 0.1 M PB on Superfrost Plus^TM microscope slides (Thermo Fisher) and coverslipped using Mowiol.