Aperio system

Tissues were fixed in 10% buffered formalin for 24 h prior to processing. Tissues were processed and embedded in paraffin and then sectioned at 5 μm. Slides were deparaffinized in several baths of xylene and then rehydrated in graded alcohols followed by ddH₂O. Slides were incubated in 1× pH 6 citrate buffer (Invitrogen, 00-5000) in DAKO PT Link for 20 min. IHC was performed using DAKO Autostainer Plus following manufacturer’s instructions. Slides were incubated in 3% H₂O₂ for 15 min. To block non-specific binding, tissues were incubated with 10% normal goat serum for 30 min, followed by avidin/biotin block (Vector Labs, SP-2001). Primary antibody LSD1 (Cell Signaling, 2139) or VDR (Thermo Scientific MA1710 (Clone 9A7)) were diluted in 1% BSA solution and incubated for 30 min at room temperature, followed by the biotinylated Goat Anti-Rabbit secondary antibody (Abcam, ab6720) for 15 min. For signal enhancement, ABC reagent (Vector Labs, PK-6100) was applied for 30 min. Slides were then incubated with DAB substrate (Dako, K3467) for 5 min and then counterstained with DAKO hematoxylin for 20 s. Slides were dehydrated through several baths of graded alcohols and xylenes and then coverslipped. Finally, slides were scanned using the Aperio System (Leica).

All TMA spots were scanned at a high resolution (20×) using the Aperio system (Aperio Technologies, Inc., Vista, CA, USA) and scored manually on computer screen independently by two pathologists (XWG and CRZ) in a blinded manner. The scoring of PTEN expression was based on the intensity and extent of staining and was evaluated according to the following histological scoring method. The mean proportion of staining cells was determined semiquantitatively and scored as follows: 0 for staining <1%, 1 for 1 to 25%, 2 for 26 to 50%, 3 for 51 to 75%, and 4 for >75% of the examined cells. Staining intensity was graded as follows: 0, negative staining; 1, weak staining; 2, moderate staining; 3, strong staining. The histological score (H-score) for each specimen was computed by the formula:
H-score = proportion score × intensity score
A total score of 0 to 12 was calculated and graded as negative (−, score: 0), weak (+, score: 1 to 4), moderate (++, score: 5 to 8) or strong (+++, score: 9 to 12). The Ki-67 LI was estimated as the percentage of Ki-67 positive cell nuclei by counting 500 to 1000 cells in the region of the tumor with the greatest density. In cases where the score difference was ≥2 for H-score and 10% for LI, the slides were re-examined and a consensus was reached by the observers. The mean score from each individual was calculated.

Tissue microarrays (HRec-Ade180Sur-03, Shanghai Outdo Biotech Co.) were constructed using formalin-fixed, paraffin-embedded carcinoma tissues matched to adjacent normal tissues collected from 90 colorectal cancer cases (cohort 2). Immunofluorescence was performed following an established protocol. Briefly, tissue microarrays were dewaxed, rehydrated through graded ethanol, and incubated with 3% hydrogen peroxide for 30 mins. Antigen retrieval was performed by heating the sections to 95 °C in 0.01 M citrate buffer (pH6.0) for 15 mins. Slides were then washed in PBS for 15 mins, treated with 10% normal horse serum for 30 mins and incubated with the primary antibodies, including Alexa Fluor 549 mouse anti-CD3 (BioLegend, San Diego, USA) and DyLight 488rabbit anti-TIGIT (BioLegend, San Diego, USA). After staining, the slides were mounted with ProLong Gold Antifade Mountant with 4’,6-diamidino-2-phenylindole (DAPI, Thermo Fisher, USA). The images were taken using the Leica Aperio System (Leica, USA).

For the analysis of chondrogenic differentiation in 3D pellet culture, paraffin sections with 3 μm thickness were stained with hematoxylin and eosin (HE) to evaluate pellet morphology, as well as with Alcian blue (pH 2.5) to evaluate glycosaminoglycans (GAGs) deposition. Expression of Ki-67 was evaluated immunohistochemically using mouse monoclonal antibody anti-Ki-67 (1:80; M7240; Dako Corporation, CA, USA). Glass slides were scanned with Aperio System (Leica Biosystems, CA, USA) at 40× and the results were descriptive.
For TEM, pellets were fixed with 1% osmium tetroxide for 1 h, dehydrated in ascending concentration of acetone and embedded in epoxy resin. Semithin sections with 0.5 μm were stained with 1% toluidine blue and scanned with Aperio System at 40× for descriptive analysis. Ultrathin sections (70 nm) were collected on copper grids and counterstained with 2% uranyl acetate and lead citrate. Images were obtained using JEM-1011 (JEOL, MA, USA). In the ultrastructure analysis, production of extracellular matrix was evaluated through the presence of collagen fibers and intra and extracellular vesicles.