Formalin-Fixed Paraffin-Embedded Organoid Immunohistochemistry

Following BME-2 dissociation, organoids were gently pelleted and fixed in 10% neutral formalin before paraffin embedding and sectioning. Paraffin embedded sections of 3.5 μm were stained by a Bond Max autostainer according to the manufacturer’s instruction (Leica Microsystems). Primary antibodies cytokeratin (AE1/AE3, 1:100, Dako), Ki67 (8D5, 1:400, Cell Signalling Technology), and p53 (D07, 1:50, Leica) were applied with negative controls as previously described^{2 (link)}.

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Price S., Bhosle S., Gonçalves E., Li X., McClurg D.P., Barthorpe S., Beck A., Hall C., Lightfoot H., Farrow L., Ansari R., Jackson D.A., Allen L., Roberts K., Beaver C., Francies H.E, & Garnett M.J. (2022). A suspension technique for efficient large-scale cancer organoid culturing and perturbation screens. Scientific Reports, 12, 5571.

Publication 2022

Bond Max cytokeratin (AE1/AE3, Ki67 (8D5,

Antibodies Cytokeratin Embedded paraffin Formalin Organoids

Corresponding Organization :

Other organizations : Wellcome Sanger Institute, University of Cambridge, MRC Cancer Unit

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Variable analysis

independent variables

BME-2 dissociation

dependent variables

Staining of cytokeratin (AE1/AE3)
Staining of Ki67 (8D5)
Staining of p53 (D07)

control variables

Paraffin embedding
Sectioning
Staining using a Bond Max autostainer
Negative controls as previously described

controls

Negative controls as previously described

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