Protocol detail

Immunohistochemical Analysis of Tumor Samples

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Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tumour tissue using antibodies against human Ki67(Cell Signaling #9027, 1:400 dilution), anti-human mitochondria (Abcam ab92824, 1:1000 dilution) and against endogenous NUT protein (Cell Signaling #3625, 1:50 dilution) used to detect NUT fusion proteins [7 (link), 10 (link)]. Digital images of whole tissue sections were acquired using a Leica SCN400 histology scanner (Leica Microsystems). Ki67-positive index was evaluated using Halo Software V. 3.2 (Indica lab). Regions of interest (ROIs) within the tissue sections were first identified using Halo Software via machine learning technology across pathological samples and tissue control, within these ROIs, nuclei were detected and classified as positive or negative based on IHC staining thresholds.

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Tontsch-Grunt U., Traexler P.E., Baum A., Musa H., Marzin K., Wang S., Trapani F., Engelhardt H, & Solca F. (2022). Therapeutic impact of BET inhibitor BI 894999 treatment: backtranslation from the clinic. British Journal of Cancer, 127(3), 577-586.

Publication 2022

ab92824 Leica SCN400 histology scanner

Antibodies Dilution Embedded paraffin Formalin Human Immunohistochemistry Mitochondria Nuclei Nut proteins Tissue Tumour

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Variable analysis

independent variables

Antibodies used for immunohistochemistry:
- Anti-human Ki67 (Cell Signaling #9027, 1:400 dilution)
- Anti-human mitochondria (Abcam ab92824, 1:1000 dilution)
- Anti-endogenous NUT protein (Cell Signaling #3625, 1:50 dilution)

dependent variables

Ki67-positive index evaluated using Halo Software V. 3.2 (Indica lab)

control variables

Formalin-fixed, paraffin-embedded tumour tissue
Whole tissue sections imaged using a Leica SCN400 histology scanner (Leica Microsystems)
Regions of interest (ROIs) within the tissue sections identified using Halo Software via machine learning technology across pathological samples and tissue control
Nuclei detected and classified as positive or negative based on IHC staining thresholds within the identified ROIs

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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