Visualisation of kupffer cells in the rat liver with poly- and monoclonal antibodies against microglial-specific protein Iba-1

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Abstract

BACKGROUND: A necessary attribute of any study, which is related to the cell biology of various structural components of the digestive tract, is the usage of modern immunohistochemical methods. One of the most difficult objects are resident liver macrophages, or Kupffer cells. This determines the high significance to develop a reliable approach for Kupffer cells visualization.

AIM: The aim of the study was to develop a protocol for the immunohistochemical research of Kupffer cells in rat liver using two types of primary antibodies against Iba-1, and to analyze the advantages and disadvantages of this approach, taking into account the use of zinc-ethanol-formaldehyde as a fixative.

MATERIALS AND METHODS: The study was carried out on liver samples of adult Wistar rats (n = 5). Goat polyclonal and rabbit monoclonal antibodies against calcium-binding protein Iba-1 were used for the ligh microscopy immunohistochemistry assay of resident liver macrophages.

RESULTS: Using two types of antibodies it was shown by quantitative analysis that rabbit monoclonal antibodies most completely reveal Iba-1-immunopositive structures compared to goat polyclonal antibodies. Fixation with zinc-ethanol-formaldehyde made it possible to reveal Iba-1-immunopositive cells in all studied rat liver samples.

CONCLUSIONS: Iba-1 immunohistochemistry using rabbit monoclonal antibodies was considered as the most optimal immunohistochemical approach. Fixation with zinc-ethanol-formaldehyde preserves the antigenic epitopes and allows the effective use of different antibodies.

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About the authors

Inga A. Nikitina

Institute of Experimental Medicine

Email: inga06819@gmail.com

Junior Research Associate, Laboratory of Experimental Histology and Confocal Microsopy, Department of General and Special Morphology

Russian Federation, Saint Petersburg

Valeria A. Razenkova

Institute of Experimental Medicine

Author for correspondence.
Email: valeriya.raz@yandex.ru
ORCID iD: 0000-0002-3997-2232
SPIN-code: 8877-8902
Scopus Author ID: 57219609984
ResearcherId: AAH-1333-2021

PhD student, Junior Research Associate, Laboratory of Functional Morphology of the Central and Peripheral Nervous System, Department of General and Special Morphology

Russian Federation, Saint Petersburg

Olga V. Kirik

Institute of Experimental Medicine

Email: olga_kirik@mail.ru
ORCID iD: 0000-0001-6113-3948
SPIN-code: 5725-8742
Scopus Author ID: 27171304100
ResearcherId: A-8710-2012

Cand. Sci. (Biol.), Senior Research Associate of the Laboratory of Functional Morphology of the Central and Peripheral Nervous System, Department of General and Special Morphology

Russian Federation, Saint Petersburg

Dmitrii E. Korzhevskii

Institute of Experimental Medicine

Email: DEK2@yandex.ru
ORCID iD: 0000-0002-2456-8165
SPIN-code: 3252-3029
Scopus Author ID: 12770589000
ResearcherId: C-2206-2012

MD, Dr. Sci. (Med.), Professor of the Russian Academy of Sciences, Head of the Laboratory of Functional Morphology of the Central and Peripheral Nervous System, Department of General and Special Morphology

Russian Federation, Saint Petersburg

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2. Fig. 1. Iba-1-immunopositive cells in rat liver: a, c — immunohistochemical reaction with polyclonal goat antibodies; b, d — reaction with monoclonal rabbit antibodies; a, b — with dyeing of hepatocyte nuclei with alum hematoxylin, microscope magnification ×40; c, d — microscope magnification ×10. The asterisk indicates the central vein

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3. Fig. 2. The result of digital transformation of images showing the clustering of Iba-1-immunopositive cells in rat liver preparations. DBSCAN algorithm, parameter value ε = 50, minDensity = 5 objects were used: a — Iba-1-immunohistochemistry with polyclonal goat antibodies; b — Iba-1-immunohistochemistry with monoclonal rabbit antibodies

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4. Fig. 3. Area occupied by Iba-1-positive structures: a — рaired difference comparison of the total staining area of Iba-1-immunopositive cells using polyclonal goat and monoclonal rabbit antibodies; *p < 0.05; b — result of statistical comparison of staining area of Iba-1-immunopositive cells for all pairs of cases (n = 5; N1–N5) for polyclonal goat and monoclonal rabbit antibodies. On the Y-axis on the left of each graph, a scale-invariant projection of the Y-axis scale on the right (µm2) on the interval from [0, 1] is located; *p < 0.05, **p < 0.01

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