Scn400 slide scanner

After microCT imaging, samples were rinsed in PBS for 2 days before being embedded in paraffin using a Vacuum Infiltration Tissue Processor (Tissue-Tek VIP, Sakura Finetek, Netherlands). Transversal sections of 5 μm thick were made using a microtome and stained with hematoxylin and eosin (H&E) for comparison with cryo-CECT. Images of the histological sections were obtained using a SCN400 Slide Scanner (Leica Microsystems, Germany).

After microCT scanning, samples were rinsed in PBS for at least 2 days and embedded in paraffin. Sections of 5 µm thick were made using a microtome and stained with hematoxylin and eosin (H&E), Masson’s trichrome or Sirius red for comparison with cryo-CECT. Histological sections were imaged using a SCN400 Slide Scanner (Leica Microsystems, Germany).

All sections were imaged at 20× magnification using a Leica SCN400 slide scanner and individual images were exported using Leica Ariol software (Leica Microsystems, Buffalo Grove, IL, USA). All images were adjusted for brightness and contrast, but were otherwise unaltered for the purposes of this study. For Figures 1, 3, and 4, images were resized relative to the largest panel (usually those depicting chimpanzee V1) to provide better comparisons and distinctions between laminar boundaries across the range of variably sized primates and tree shrews. For Figures 2 and 5, all images were held at absolute size to display the relative thickness of V1 layers compared to total cortical thickness in Figure 2, as well as the relative neuronal densities between layers of V1 and V2 in Figure 5.
For comparative estimates of neuronal density, roughly 100–120 50 μm² regions were delineated as part of layer 3 or layer 4 in V1 or V2 on NeuN-stained sections from each species, and the number of stained cells in each region was automatically derived using count functions in Ariol. Raw counts were analyzed using the Kolmogorov–Smirnov test and were determined to be non-normal (p < 0.0001). Significant differences below p = 0.05 were then determined using the Mann–Whitney U test in IBM-SPSS (v. 22; IBM, Armonk, NY, USA).

Briefly, mouse brains were fixed by intracardiac perfusion with 4 % paraformaldehyde in phosphate-buffered saline (PBS) and postfixation in the same buffer. Specimens were then embedded in paraffin, and sections processed for immunohistochemical staining with antibody against TH (1:100) or dopamine D2 receptor (1:50, Biorbyt Ltd, Cambridge, UK). Slides were scanned at ×20 using a Leica SCN400 slide scanner (Leica Microsystems Inc., Buffalo Grove, IL). Immunofluorescent staining was carried out with dopamine D2 receptor, and the slides were imaged at ×20 using a Leica DMI3000 B.

Formalin-fixed, paraffin-embedded tissue sections (4 μm) were deparaffinized by incubating slides at 60 °C for 1 h followed by repeated xylene and ethanol submersion. Antigen retrieval was performed with Diva Decloaker 10X (Cedarlane, ON, CA), steaming for 30 min, rinsed with dH₂O and then incubated with 3% hydrogen peroxide (Sigma-aldrich, MO, USA). Sections were incubated with blocking from Vectastain ELITE ABC-Peroxidase kit according to manufacture’s protocol (Vector Laboratories, CA, USA). Slides were stained with rabbit monoclonal anti-ABCB-1 (1:100) and anti-YB-1 (1:500) antibodies overnight at 4 °C followed by secondary antibody staining using manufacturer’s protocol. Images were taken using SCN400 Slide Scanner (Leica Microsystems). Staining intensity was scored by a certified pathologist who was blinded to this study (score of 0–3). Cancer cells with positive staining in the tumor region were assigned an estimated percentage. Final intensity was calculated as: intensity = (score)×(percentage area)/100.

Protein expression of DPYSL5 was assessed in situ with immunohistochemistry. The automated staining platform DISCOVERY ULTRA (Ventana Medical Systems) was used. Briefly, antigen retrieval was performed with Cell Conditioning 1 (CC1) (Ventana) at 95^oC for 64 min. Slides were incubated with DPYSL5 antibody (CR-3, MA3-700, monoclonal, 1:200, ThermoFisher Scientific) at room temperature for 1 h. Slides were then incubated with AffiniPure Rabbit AntiRat IgG (H + L) (312-005-045, polyclonal, 1:1000, Jackson ImmunoResearch Laboratories) at 37^oC for 32 min. To visualize bound antibodies, UltraMap anti-Rb HRP and the ChromoMap DAB kit (Ventana) were used. Digital images of all immunohistochemically stained slides were acquired with SCN400 Slide Scanner (Leica Microsystems). To analyze protein expression, digital scans of stained tissue were scored with Aperio ImageScope (Leica Biosystems) by an experienced pathologist (LF).