Immunolabeling of leptin in the liver of Nile tilapia using the immunohistochemistry techniqueRESUMO
O objetivo deste estudo foi avaliar e padronizar a imunomarcação celular de leptina nos ácinos hepáticos da tilápia-do-nilo (Oreochromis niloticus), pela técnica de imuno-histoquímica (IHQ). Para a padronização da técnica de IHQ, foram obtidas amostras de fígado de alevinos de tilápia-do-nilo de 102,10 ± 18,40g. Após o preparo das lâminas por processamento histológico clássico, foi aplicada a técnica de IHQ. Anticorpos policlonais primários antileptina e bloqueador da leptina foram instilados sobre os cortes das lâminas para, depois, serem incubados em câmara úmida overnight. Posteriormente, o kit contendo o anticorpo secundário foi instilado, e as lâminas incubadas em temperatura ambiente. Por fim, os cortes foram contracorados com hematoxilina de Mayer filtrada, após adição do corante DAB. Os ácinos hepáticos foram fotografados e analisados por meio do programa Image J®. A técnica de IHQ permitiu a observação da imunomarcação específica da leptina nos ácinos hepáticos de tilápias-do-nilo, confirmada pelo bloqueio específico da marcação de leptina com o anticorpo inibidor, que obteve o mesmo comportamento que o controle negativo, no qual não foi instilado o anticorpo primário antileptina. Assim, pode-se inferir que a IHQ é uma técnica que pode ser utilizada para estudos futuros sobre fisiologia e nutrição que envolva o hormônio leptina para essa espécie de peixe.
Palavras-chave:
peixes; metabolismo; nutrição; fígado
Keywords:
fish; methodology validation; metabolism; liver
Palavras-chave:
peixes; metabolismo; nutrição; fígado
Keywords:
fish; methodology validation; metabolism; liver
Palavras-chave:
peixes; metabolismo; nutrição; fígado
Leptin (LEP) is a peptide hormone originally identified in obese rats. It participates in the signaling pathway of adipose tissue that regulates the proportion of body fat deposits and exerts endocrine, paracrine, and autocrine effects in different tissues, primarily the liver (Zhang et al., 1994). This peptide hormone plays an important role not only in controlling food intake and energy balance but also in reproductive processes and stress response in fish (Chen et al., 2020). The decrease in leptin gene expression during starvation suggests that the hormone may regulate energy homeostasis by reducing metabolic energy demand (Dar et al., 2018).
In aquaculture, nutrition plays a significant role, as about 70% of the costs of this activity are attributed to feed expenses (Passinato et al., 2015). In this context, it is important to develop techniques that provide results enabling the understanding of the interaction between nutrition and animal physiology to improve feed efficiency by enhancing the health aspects of cultured animals (Chen et al., 2020).
The liver is an important organ for metabolic and immunological regulation, playing a systemic and local role, in addition to essential functions such as metabolizing substances present in the blood (Castro et al., 2014). Fish are susceptible to environmental variations and respond more sensitively to these stimuli compared to mammals. Thus, the fish liver is considered a key organ for assessing the impact of environmental, health, and nutritional factors on the physiology and metabolism of these animals (Datta-Munshi and Dutta, 1996).
Chen et al. (2020) evaluated the expression of the leptin gene in different tissues in Yangtze sturgeon, Acipenser dabryanu, and observed that it is predominantly expressed in the liver and pancreas. The modulation of leptin gene expression is not only related to food intake but also to the action of different inflammatory mediators since leptin levels are inversely correlated with glucocorticoid hormones and increase during inflammatory, infectious, or septicemic processes through the action of cytokines such as TNF-α, IL-6, and IL-1β, as well as activation caused by lipopolysaccharide (LPS) present in Gram-negative bacteria (Procaccini et al., 2017). Through direct regulation of hepatocytes, leptin can regulate the growth axis by stimulating the expression of insulin-like growth factor 1 (IGF-1) and increasing growth hormone (GH) signaling (Won et al., 2016).
Therefore, the aim of this study was to evaluate and standardize the cellular immunolabeling of leptin in the hepatopancreatic tissue of Nile tilapia, Oreochromis niloticus, using the immunohistochemistry (IHC) technique. The procedures proposed in this study were approved by the Ethics Committee on the use of Animal (CEUA), under protocol number 202/2017.
For the standardization of the IHC technique, liver samples were obtained from three healthy Nile tilapia fingerlings weighing 102.10±18.40g, reared in a recirculating system with constant aeration. The samples were stored in jars containing 10% buffered formalin solution for 24 hours. Subsequently, tissue dehydration with alcohol, clarification with xylene, and embedding in paraffin were performed, characterizing the classic routine histological processing. The samples were sectioned at 3 μm thickness using a manual microtome (RM2245, Leica Biosystems®, San Diego, CA, USA) and fixed on slides previously treated with 3% organosilane solution (Sigma Chemical Company®, St. Louis, MO, USA) and ethanol (preparation solution 225mL of ethanol added to 25mL of organosilane).
The tissues were deparaffinized in an oven at 37°C for 30 minutes, clarified in xylene I and II for 30 minutes each, hydrated with graded alcohol from 100% to 85%, and distilled water for 10 minutes each. The histological sections were delimited with a hydrophobic pen (Liquid Blocker, Erviegas®, SP, BR). Endogenous peroxidase blocking was performed using a commercial kit (Spring Bioscence, Pleasanton, USA) instilled on the slides for 15 minutes in a humid chamber at room temperature. For antigen retrieval under humid heat conditions, after successive washes with phosphate buffer (PBS) pH 7.4, the slides were submerged in 2.4% citrate buffer solution (pH 6.0) in an aluminum pressure cooker with a capacity of 4.5 liters for 3 minutes after boiling. Immediately after removing the cooker, the slides were slowly cooled in running water and then in PBS for 5 minutes.
After successive washes with PBS, primary polyclonal antibodies (Santa Cruz Biotechnology®, CA, USA) directed against the proteins of interest for leptin (LEP): anti-leptin [Ob antibody A-20, sc-842-rabbit (IgG)]; and leptin blocking (Ob peptide blocking Z-1, 515963-P) were instilled on the tissues. The dilutions tested for the LEP antibody were 1:500, 1:1000, and 1:2000 (Catalog Ob antibody, Santa Cruz Biotechnology®, CA, USA). The dilution of 1:1000 resulted in the best immunolabeling results and was the one selected for the protocol validation. The selected dilution of the leptin-blocking antibody was also 1:1000. For the methodology validation, LEP antibody was instilled on the three slides containing the tissue sections (two per slide) of three different fingerlings, and on three other slides containing the same sections of the same fishes, the LEP antibody was instilled and then the LEP blocking peptide was added, both with negative controls (without LEP antibody addition) on the same slide. The slides were then incubated in a humid chamber at 4°C overnight.
The next day, successive washes with PBS pH 7.4 were performed, and then the secondary antibody was instilled using the Reveal Polyvalent HRP® Kit (Spring Bioscience, Pleasanton, CA, USA) on the slides incubated in a covered humid chamber for 30 minutes at room temperature. Afterward, the reaction was revealed by adding a diaminobenzidine-peroxidase (DAB-K3468, DAKO Corporation®, Carpinteria, CA, USA) solution, added for a standard time of 2 minutes for all sections. The slides were washed in distilled water and counterstained with Mayer's hematoxylin (Merck®, Darmstadt, Germany) for 1 minute, then washed in running water for 10 minutes and dehydrated in decreasing alcohol gradients, clarified in decreasing xylene concentrations, and mounted with synthetic resin (Sigma-Aldrich® Chemical Company, St. Louis, USA) on coverslips. LEP immunolabeling, LEP blocker, and negative control were analyzed under a bright-field microscope (Olympus BX 51, Tokyo, Japan) with a 10X objective lens coupled to a camera.
The results of the immunolabeling using LEP primary antibody, LEP blocker _ LEP primary antibody and their respective negative controls are presented in Fig. 1. LEP immunolabeling was more intense in the pancreatic acini, (Fig. 1 A, D and G). This result agrees to that found in the study by Pacheco et al. (2021), which used the same LEP antibody in the hepatopancreatic tissue of Nile tilapia, demonstrating that the leptin hormone has a specific action in this organ and that local concentration is affected by fasting time for this fish species.
Immunolabeling for leptin in the hepatopancreatic tissues of Nile tilapia, Oreochromis niloticus. Letters A, D, and G: histological sections from three different animals incubated with the primary anti-leptin antibody. Letters B, E, and H: negative controls, using the histological sections from the same three different animals at the same portion of the tissue. Letters C, F, and I: histological sections from the same three different animals at the same portion of the tissue, containing the LEP antibody with the addition of the LEP blocking peptide. Photomicrograph at 10 X magnification.
The IHC technique used proved effective in verifying the immunolabeling of the hepatopancreatic tissue of Nile tilapia, highlighting the presence of leptin in this tissue as described by other authors (Angotzi et al., 2016; Li et al., 2019; Chen et al., 2020; Pacheco et al., 2021).
Thus, the IHC technique allowed specific labeling of leptin in the acini of the hepatopancreatic tissue of Nile tilapia, demonstrating its efficiency for use in future studies involving the nutrition, physiology, and metabolism of this hormone in this fish species.
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Publication Dates
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Publication in this collection
27 Jan 2025 -
Date of issue
Jan-Feb 2025
History
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Received
14 June 2024 -
Accepted
23 July 2024
