Organoid Immunohistochemistry on Ventana Benchmark XT

Fix organoids in 10% NBF for 15-30 minutes.

Prepare paraffin-embedded blocks on tissue processer.

Cut 5μm sections from FFPE (formalin-fixed paraffin-embedded) blocks using microtome.

Mount sections on positively charged slides and bake overnight at 70° C.

Prepare primary antibody dilution kit (depending on antibody concentration) for Ventana Benchmark XT automatic staining platform (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol.

Load slides onto Ventana and run through alcohol, antigen retrieval, primary antibody incubation and visualization steps.

Coverslip slides and let dry overnight.

Scan slides at 40x on Aperio VERSA 8 (Leica Biosystems, Wetzlar Germany) digital slide scanner.

Manually segment organoids to generate annotatios in QuPath (version 0.2.3,https://QuPath.github.io/).

Perform color deconvolution and determine appropriate settings for positive-pixel-based analysis.