Color deconvolution algorithm

Slides were digitized using a high-resolution whole-slide digital ScanScope scanner (Aperio Technologies, Inc.). The digital images from hematoxylin and eosin-stained slides were viewed and quantified using the tools of ImageJ software. The whole implant was contoured to acquire the total implant area in pixels (TA). All areas of bone are selected to obtain total bone area in pixels (BA). The BA/TA ratio was calculated and reported as percentage (n = 3 sections per implant and 4 implants/condition). The digital images from Sirius red-stained slides were viewed and analyzed using Aperio's viewing and image analysis tools. In each slide, five rectangular fields with a fixed area of 1.18 mm² (total analysis area) were randomly selected. A color deconvolution algorithm (Aperio Technologies, Inc.) was employed to measure areas of the red color of stained collagen (positive staining of Sirius red), and its percentage relative to the total area was calculated (n = 4 implants per treatment).

Slides were scanned at 20X magnification using a ScanScope XT slide scanner (Aperio Technologies, Vista, CA, USA). The Spectrum Analysis algorithm package, ImageScope analysis software and Color Deconvolution algorithm (version 9; Aperio Technologies.) were applied to quantify immunohistochemical (IHC) staining. These algorithms were used to calculate the average positive intensity (API), as well as the area of positive staining, and the percentage of weak (1+), medium (2+), and strong (3+) positive staining. The final API was subtracted from 255, as these intensity ranges on an 8-bit scale of 0 to 255 (black to white, respectively). The maximum value from both cores for each patient was used for statistical analysis.

The obtained liver tissues via liver biopsy were fixed in 10% formalin (Sigma). Tissue samples were embedded in paraffin blocks and then sliced into 5-μm-thick sections. Sections were processed and stained with Masson’s trichrome as reported [21 (link)]. Masson staining kits were from Abcam Co., Ltd. (Trichrome Stain, ab150686). Collagen stained blue (Additional file 2: Figure S2). In order to characterize collagen area, Masson’s trichrome-stained slides were scanned with a Leica SCN400 scanner (Leica Microsystems) at × 40 magnification and measured using Aperio ImageScope (v12.3.2.5030, Aperio Technologies). The images were saved as “.scn” format files. The Color Deconvolution algorithm (v9, Aperio Technologies) was used to isolate individual stains for semi-quantification. The percent total positive, total stained area (mm²), and total analysis area (mm²) in each visual field were measured and recorded. The analytical data were saved as “.xls” format files. CPA = percent total positive × total stained area/total analysis area.