Immunohistochemistry Analysis of Ki67 Expression

Formalin-fixed and paraffin-embedded specimens were used for immunohistochemistry staining. Six baseline samples and four after-treatment samples were included. Fewer after-treatment samples were included due to sample loss. For each sample, three replicates were prepared. 5 μm sections of paraffin-embedded endometrial tissues were prepared and immunohistochemistry staining was performed using a standardized protocol (25 (link)). Primary antibody against Ki67 (catalog no. NB110-89717, Novus biologicals, Biotechne, USA) were diluted in 1:200 using diluent DaVinci Green (Biocare Medical, Concord, CA) and incubated overnight at 4 °C. Rabbit MACH 3TM Probe and its respective HRP polymer (Biocare Medical, Concord, CA) and Betazoid DAB Chromogen (Biocare Medical, Concord, CA) were used to detect the antibody. Finally, tissue sections were counterstained using hematoxylin (Vector Laboratories, Inc., Burlingame, CA) and mounted using the xylene-based medium Pertex^® (Histolab, Gothenburg, Sweden). Immunopositivity for Ki67 stained areas (six randomly selected areas per slide) was analyzed at 20x magnification using Image J software.

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Tang Y., Ponandai-srinivasan S., Frisendahl C., Andersson J.K., Pavone D., Stewart E.A., Lalitkumar P.G., Korsching E., Bogavarappu N.R, & Gemzell-Danielsson K. (2023). Bromocriptine inhibits proliferation in the endometrium from women with adenomyosis. Frontiers in Endocrinology, 14, 1026168.

Publication 2023

Betazoid DAB Chromogen hematoxylin

After treatment Antibody Biologicals Chromogen Embedded paraffin Endometrial Formalin Hematoxylin Mach Novus Paraffin Polymer Rabbit Tissue Vector Xylene

Corresponding Organization : Karolinska University Hospital

Other organizations : Mayo Clinic, WinnMed, University Hospital Münster, University of Münster

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Variable analysis

independent variables

Treatment (baseline vs. after-treatment)

dependent variables

Ki67 immunopositivity (stained areas)

control variables

Tissue preparation (formalin-fixed and paraffin-embedded)
Number of replicates per sample (three)
Immunohistochemistry staining protocol (standardized)
Primary antibody (Ki67)
Antibody dilution (1:200)
Incubation conditions (overnight at 4 °C)
Detection system (Rabbit MACH 3TM Probe, HRP polymer, and Betazoid DAB Chromogen)
Counterstaining (hematoxylin)
Mounting medium (Pertex®)

controls

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

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