Nov 09, 2022
Organoid Immunohistochemistry on Ventana Benchmark XT
Forked from a private protocol
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: gustavo.parfitt Parfitt, kristen.whitney 2022. Organoid Immunohistochemistry on Ventana Benchmark XT. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj88kpgk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 09, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 72539
Keywords: ASAPCRN
Funders Acknowledgement:
Kristen Whitney
Abstract
Organoid Immunohistochemistry on Ventana Benchmark XT
Immunohistochemistry of organoid sections
Immunohistochemistry of organoid sections
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.
Digital Histopathologic Analysis in QuPath
Digital Histopathologic Analysis in QuPath
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.