Mach2 double stain 1

Serial 4-μm-thick paraffin sections from formalin-fixed, paraffin-embedded xenografts, obtained as described above (see In vivo studies) and as previously reported (8, 11 (link)), were processed according to standardized IHC procedures and then immunostained with the following antibodies: anti-CD47 (clone EPR21794; #ab218810, rabbit monoclonal, Abcam), anti-F4/80, (D2S9R) (#70076, rabbit polyclonal, Cell Signaling, RRID: AB_2799771), anti-CD206 (#ab64693, rabbit polyclonal, Abcam, RRID: AB_1523910), and anti-CD99 (O13) (#915601; BioLegend, RRID:AB_2565169). An avidin–biotin–HRP method was used for single staining (VECTASTAIN ABC kit, PK-4001, Vector Laboratories). A polymer with anti-mouse alkaline phosphatase and anti-rabbit HRP was used for dual staining (Mach2 Double Stain #1, #PBC-MRCT523L, Biocare Medical). For morphologic analyses, the slides were stained with hematoxylin and eosin.
Histologic and histomorphometric analyses were carried out with a digital pathology slide scanner with a resolution of 0.5 μm/pixel (Aperio AT2, Aperio Technologies, Vista, CA) using ImageScope software (v12.4.3, RRID: SCR_014311) for slide viewing and analysis.

2-μm-thick consecutive tissue sections were prepared from formalin-fixed and paraffin-embedded tissues, provided by the Pathology Department of the Humanitas Clinical and Research Center, and processed for immunohistochemistry. Briefly, after deparaffinization and rehydration, antigen retrieval was performed by heat treatment using EDTA buffer (Dako; 0.25 mM, pH 8) in water bath at 98°C for 20 min. Endogenous peroxidases were blocked by incubation with Peroxidase-Blocking Solution for 15 min at room temperature, followed by incubation for 20 min with Background Sniper (Biocare Medical) to block nonspecific binding. The sections were then incubated with primary antibodies anti-human CD68 (Dako; KP-1 clone, diluted 1:1,000), CD163 (Leica Biosystems, 10D6 clone, diluted 1:200) for 1 h at room temperature, followed by incubation with the detection system EnVision+System HRP-labeled anti-mouse (Dako). Diaminobenzidine tetrahydrochloride (Biocare Medical) was used as chromogen. Nuclei were lightly counterstained with a freshly made hematoxylin solution (Dako). LXR staining was performed with anti-human LXRα antibody (LSBio; polyclonal, diluted 1:100), together with anti-CD163, followed by incubation with the detection system Mach2 Double Stain 1 (Biocare Medical). Ferangi Blue (Biocare Medical) and 3,3′-Diaminobenzidine were used as chromogens.

Formalin-fixed, paraffin-embedded tissues were cut at 5 μm thickness. We used the BenchMark XT automated slide staining system (Ventana Medical Systems Inc.) to optimize antibody dilutions and staining. The antigen retrieval step (basic pH tris base buffer) was performed using the Ventana CC1 solution. Primary antibody incubation of sections was for 1 hour. The Ventana iView DAB detection kit was used as the chromogen, and the slides were counterstained with hematoxylin.
For double-IHC staining, the BenchMark XT automated slide staining system (Ventana Medical Systems Inc.) was used for deparaffinization and antigen retrieval. The antigen retrieval step was performed using the Ventana CC1 solution, which is a basic pH tris base buffer. Tissue was incubated with the antibody cocktail for 1 hour at 37°C. Tissue was then incubated with mouse AP (alkaline phosphatase) + rabbit horseradish peroxidase (HRP) polymer detection kit (Biocare Mach 2 Double stain 1) for 30 min at RT. Tissues were rinsed with tris-buffered saline and incubated with chromogens Betazoid DAB and Warp Red (both Biocare) for 5 min each, respectively. Slides were counterstained with hematoxylin, dehydrated, cleared, and coverslipped. The Aperio VERSA 8 slide scanning system from Leica Biosystems, equipped with a Point Grey Grasshopper3 color camera for bright-field scanning, was used to analyze stained sections.