Vslide

Immediately after liver and spleen harvest, macroscopic evaluation was performed and the spleen was weighted. Samples of the liver and spleen were fixed using a 4% formaldehyde solution (Merk - Darmstadt, Germany) in Millonig's buffer (0.1 M sodium hydroxide, 0.13 M sodium phosphate monobasic—Sigma-Aldrich) and later processed for histology. Paraffin-embedded sections (5 μm) were stained with hematoxylin and eosin (HE), Giemsa or Prussian blue. The slides were analyzed with the aid of a microscope (AxioImager A2, Zeiss, Oberkochen, Germany) and images were captured using an Axiocam HRM (Zeiss) and the software AxionVision Release 4.8.2 (Zeiss).
For hemozoin quantitation in the spleen, the histology slices were dyed with Nuclear Fast Red and mounted with coverslips. Each slide was then completely scanned using VSlide (Metasystems, Germany), generating high definition images (0.8 NA) of all the slices. Because the original images were too big, they were divided into three parts for analysis, by using ImageJ, where the red (nuclei) and brown/black (hemozoin) colors were separated with the color threshold function. The hemozoin areas and the total tissue area in each image were measured in square pixels. These results were then used to estimate the relative hemozoin area in each slide.

The specificity of SATB1 and SATB2 antibodies was further evaluated in immunohistochemical experiments.
Tissue sections (4 μm) were cut from TMAs containing 18 normal (fallopian tube, cervix, endometrium, placenta, testis, prostate, liver, pancreas, rectum, colon, stomach, duodenum, small intestine, cerebellum, cerebral cortex, skin, skeletal muscle, and tonsil) and 7 cancer (prostate, colorectal, ventricular, renal, liver, lung, and breast) tissues. Prior to immunostaining, the sections were baked at 50 °C overnight and deparaffinized in xylene and graded ethanol. Antigen retrieval was then performed using citrate buffer pH 6 (ThermoFisher Scientific, Waltham, MA, USA) in decloaking chamber (Biocare Medical, Walnut Creek, CA, USA). Sections were stained with anti-SATB1rabbit monoclonal antibody (Clone EPR3895, Epitomics, Burlingame, CA, USA) diluted 1:100 or mouse monoclonal antibody against SATB2 (AMAb90679, CL0320, Atlas Antibodies, Stockholm, Sweden) diluted 1:1,000 in Autostainer 480S (ThermoFisher Scientific, Waltham, MA, USA) using a commercial kit (UltraVision LP HRP polymer®, Primary Antibody Enhancer, Ultra V Block and DAB plus substrate system®, ThermoFisher Scientific, Waltham, MA, USA). Slides were counterstained with hematoxylin and mounted using Pertex.
Slides were examined, and images were taken using an automated system (VSlide, Metasystems).