Aperio cs2 digital pathology slide scanner

An automated TM-Sprayer (HTX Technologies,
LLC, Chapel Hill, USA) was used for the application of the lipid standard
between 0.01 and 0.1 g/L concentrations in 2:1 CHCl₃:MeOH
onto clean ITO slides for 1 to 10 layers using the following protocol:
spray flow rate 0.12 mL/min, 30 s drying time between
layers, velocity of 1200 mm/min, 3 mm track spacing, at 30 °C.
This created concentrations from 0.33 to 3.3 ng/mm² (assuming
equal dispersion). Samples were then treated equivalently to brain
tissue sections, where 15 layers of 7 mg/mL norharmane matrix in
2:1 CHCl₃:MeOH were sprayed using the TM-Sprayer using
the same settings used for the lipid standards.
Hematoxylin
and eosin (H&E) staining was performed after MALDI imaging. Matrix
was removed from tissue by immersion in 70% ethanol for 3 min. A standard
H&E protocol was then used (70% EtOH, Milli-Q, hematoxylin, running
tap water for 3 min; eosin for 30 s; running tap water for 3 min;
100% EtOH for 1 min; xylene for 30 s; covered with a coverslip using
Entellan mounting medium). High-resolution optical images of stained
tissues were generated using an Aperio CS2 digital pathology slide
scanner (Leica Biosystems, Wetzlar, Germany). Annotations of the rat
brain are shown in Figure S1.

Animals were euthanized by CO₂ exposure followed by thoracotomy. Carcasses were kept on ice until tissue processing was completed. Lungs were excised, and the right lung was ligated, removed, and flash frozen in liquid nitrogen for protein and RNA analysis. The left lung was inflation fixed with 4% paraformaldehyde instilled through the trachea at 20 cm pressure for 2 minutes. After 24 hours, tissues were transferred to 70% ethanol prior to histologic analysis. Lungs were cut into four levels and oriented with alternating surfaces exposed prior to creation of 5 μm paraffin embedded sections. The heart was excised and the apex flash frozen, the remaining heart tissue was fixed in 10% neutral buffered formalin. The atria of the hearts were removed, and the remaining tissue oriented such that transverse sections of the ventricles could be obtained after paraffin embedding. All histological sectioning and staining were performed by UTHSCSA's Pathology Laboratory.
Lung slides were stained with hematoxylin and eosin stain, von Willebrand Factor, Masson's Trichrome, and α‐smooth muscle actin. Digital slides were created using the Aperio CS2 Digital Pathology Slide Scanner (Leica Biosystems, Wetzlar, Germany). Measurements were made using the following software: Aperio ImageScope software version 12.3.3, Pathomation (Belgium), MIPCloud (Iran), Image J, and Digimizer (Belgium).

Sections of tibiae (with tumors) were deparaffinized in xylene and degraded alcohols. Heat-induced epitope retrieval was performed by using a 2100-Retriever. Slides were rinsed with PBS, and a hydrophobic barrier was created around the tissue using a hydrophobic barrier pen (Vector Laboratories, H-4000-2). Then, slides were placed in an incubating chamber with blocking solution (Vector Laboratories, SP-6000) for 10 min and rinsed with PBS, followed by incubation with 20% horse serum (Vector Laboratories, PK-7200) for 20 min. Next, slides were incubated with the primary antibody against RFP (1:400, Abcam, ab62341, RRID: AB_945213) at 4 °C overnight and rinsed with PBS, followed by incubation with a Horse Anti-Rabbit IgG Antibody (H + L), Biotinylated, R.T.U. (Vector Laboratories, BP-1100-50) or goat IgG HRP-conjugated antibody (R&D systems, HAF017) for 30 min. After being washed again with PBS, slides were incubated with the avidin-biotin detection complex (ABC; Vector Laboratories, SK-4100) for 30 min and were then developed with 3,3′-diaminobenzidine (DAB) solution (Vector Laboratories, SK-4100). Counterstaining was performed by using Hematoxylin QS (Vector Laboratories, H-3404). Slides were scanned with an Aperio CS2 Digital Pathology Slide Scanner (Leica Biosystems).