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Establishment of primary cell cultures from canine mammary gland malignant tumours: a preliminary study

Journal of Veterinary Research
Volume 69 (2025): Issue 1 (March 2025)
By:
Open Access
|Mar 2025

Figures & Tables

Fig. 1.

Microphotographs of mammary gland tumours. A – magnification 200×, scale bar 50 μm; B – magnification 400×, scale bar 20 μm
Microphotographs of mammary gland tumours. A – magnification 200×, scale bar 50 μm; B – magnification 400×, scale bar 20 μm

Fig. 2.

Primary cell cultures derived from mammary gland solid adenocarcinoma and carcinosarcoma. A and G – cellular debris and blood cells present directly after plating the cells on day in vitro (DIV) 0; B and H – cells before washing on DIV 1; C and I – cells after washing on DIV 1. Washing the cells effectively removed the majority of cellular debris and non-adherent cells; D and J – enlarged irregular and star-shaped cells on DIV 3; E and K – larger polygonal and irregularly shaped cells on DIV 5; F and L – cells with a range of morphologies from spindle-shaped to polygonal-shaped on DIV 7. Magnification 200×, scale bar 50 μm
Primary cell cultures derived from mammary gland solid adenocarcinoma and carcinosarcoma. A and G – cellular debris and blood cells present directly after plating the cells on day in vitro (DIV) 0; B and H – cells before washing on DIV 1; C and I – cells after washing on DIV 1. Washing the cells effectively removed the majority of cellular debris and non-adherent cells; D and J – enlarged irregular and star-shaped cells on DIV 3; E and K – larger polygonal and irregularly shaped cells on DIV 5; F and L – cells with a range of morphologies from spindle-shaped to polygonal-shaped on DIV 7. Magnification 200×, scale bar 50 μm

Fig. 3.

Immunofluorescence staining to show mucin 1 (MUC1), cytokeratins 8 and 18 (CK8/18) and Kiel 67 (Ki-67) expression in canine mammary gland tumour cells. A and B – solid adenocarcinoma-derived and carcinosarcoma-derived cells expressing MUC1; C – solid adenocarcinoma-derived cells showing higher expression of CK8/18 than carcinosarcoma-derived cells; D – carcinosarcoma-derived cells showing lower expression of CK8/18 than solid adenocarcinoma-derived cells; E – solid adenocarcinoma-derived cells showing lower expression of Ki-67 than carcinosarcoma-derived cells; F – carcinosarcoma-derived cells showing higher expression of Ki-67 than solid adenocarcinoma-derived cells; G and H – 400× magnification of details of E and F. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Magnification 100×, scale bar 100 μm
Immunofluorescence staining to show mucin 1 (MUC1), cytokeratins 8 and 18 (CK8/18) and Kiel 67 (Ki-67) expression in canine mammary gland tumour cells. A and B – solid adenocarcinoma-derived and carcinosarcoma-derived cells expressing MUC1; C – solid adenocarcinoma-derived cells showing higher expression of CK8/18 than carcinosarcoma-derived cells; D – carcinosarcoma-derived cells showing lower expression of CK8/18 than solid adenocarcinoma-derived cells; E – solid adenocarcinoma-derived cells showing lower expression of Ki-67 than carcinosarcoma-derived cells; F – carcinosarcoma-derived cells showing higher expression of Ki-67 than solid adenocarcinoma-derived cells; G and H – 400× magnification of details of E and F. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Magnification 100×, scale bar 100 μm

Fig. 4.

Immunocytochemistry controls verifying the specificity of primary antibody staining. A – non-cancerous cells isolated from canine mammary glands demonstrating weak positive staining for mucin 1 (MUC1) (I) and cytokeratins 8 and 18 (CK8/18) (II), and minimal Kiel 67 (Ki-67) staining (III). (IV) is a 400× magnification of details of (III); B – negative controls where primary antibodies (MUC1 (I), CK8/18 (II) and Ki-67 (III)) were excluded from the assay. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Magnification ×100, scale bar 100 μm
Immunocytochemistry controls verifying the specificity of primary antibody staining. A – non-cancerous cells isolated from canine mammary glands demonstrating weak positive staining for mucin 1 (MUC1) (I) and cytokeratins 8 and 18 (CK8/18) (II), and minimal Kiel 67 (Ki-67) staining (III). (IV) is a 400× magnification of details of (III); B – negative controls where primary antibodies (MUC1 (I), CK8/18 (II) and Ki-67 (III)) were excluded from the assay. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Magnification ×100, scale bar 100 μm

Specific primary and secondary antibodies used for immunocytochemistry

Primary antibodies
MarkerPrimary antibodyType, cloneHost, isotypeReactivityDilutionCatalogue No.Supplier
mucin 1MUC1mAb, MUC1/955mouse, IgGhuman, mouse1 : 150NBP2-44658Novus Biologicals, Centennial, CO, USA
cytokeratin 8 cytokeratin 18Cytokeratins 8/18mAb, 5D3mouse, IgGhuman, mouse1 : 100NB120-17139Novus Biologicals
Ki-67Anti-Ki-67pAb, N/Arabbit, IgGhuman, mouse1 : 300ab15580Abcam, Cambridge, UK
DOI: https://doi.org/10.2478/jvetres-2025-0007 | Journal eISSN: 2450-8608
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Language: English
Page range: 159 - 168
Submitted on: Jul 17, 2024
Accepted on: Feb 10, 2025
Published on: Mar 1, 2025
Published by: National Veterinary Research Institute in Pulawy
In partnership with: Paradigm Publishing Services
Publication frequency: 4 times per year
Keywords:
canine mammary gland tumour,
primary cell culture,
MUC1,
CK8/18,
Ki-67
Related subjects:
Life sciences,
Molecular biology,
Microbiology and virology,
Life sciences, other,
Medicine,
Veterinary medicine

© 2025 Patŕicia Petrouškova, Nikola Hudáková, Viera Almášiová, Alexandra Valenčáková, L’ubica Horňáková, Mykhailo Huniadi, Daša Čížková, published by National Veterinary Research Institute in Pulawy
This work is licensed under the Creative Commons Attribution 4.0 License.

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