Protocol detail

Organoid Immunohistochemistry Protocol

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Organoids were fixed in 4% paraformaldehyde overnight at 4 °C and resuspended in 2% low-melting agarose prior to paraffin embedding. 4-µm sections were cut and processed for immunohistochemistry analysis or Hematoxylin and Eosin (H&E) staining as previously reported.^{24 (link)} The following primary antibodies were used: active caspase 3 (R&D systems, Abingdon, UK), Chromogranin A and Ki-67 (Abcam, Cambridge, UK), Mucin 2 (Santa Cruz Biotechnology, Heidelberg, Germany), and Lysozyme (Dako, Cambridge, UK).

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Lorenzi F., Babaei-Jadidi R., Sheard J., Spencer-Dene B, & Nateri A.S. (2016). Fbxw7-associated drug resistance is reversed by induction of terminal differentiation in murine intestinal organoid culture. Molecular Therapy. Methods & Clinical Development, 3, 16024-.

Publication 2016

active caspase 3 Mucin 2 Lysozyme

Agarose Antibodies Caspase 3 Chromogranin a Eosin Hematoxylin Immunohistochemistry Lysozyme Mucin 2 Organoids Paraformaldehyde

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Variable analysis

independent variables

Paraformaldehyde concentration (4%)
Incubation duration (overnight)
Incubation temperature (4 °C)
Agarose concentration (2% low-melting)
Paraffin embedding

dependent variables

Expression of active caspase 3
Expression of Chromogranin A
Expression of Ki-67
Expression of Mucin 2
Expression of Lysozyme
Histological analysis (Hematoxylin and Eosin staining)

control variables

Organoid culture conditions
Experimental protocol (as previously reported in reference 24)

positive controls

Not specified

negative controls

Not specified

Annotations

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