Abstract
Investigation and studies of pulmonary diseases and injuries require pre-clinical animal models. The rabbit lung model is widely used and allows for a diverse set of readouts. Among them, histology and immunohistochemistry are of invaluable merit because qualitative and quantitative information about tissue morphology and composition can be easily obtained. In this technical note, we performed several histological and immunohistochemical stainings in the rabbit healthy naïve lung tissue. Overnight formalin fixation with subsequent paraffin embedding was compared to cryopreservation with a subsequent 10-minute formalin fixation prior to staining. Antigen retrieval (AR) for paraffin embedded sections proved to enhance the corresponding signals compared to analogous staining without AR. Advantages and disadvantages of chromogenic versus immunofluorescence stainings were discussed. In addition, several morphological structures, such as the intrapulmonary bronchus with its mucosal folds, the pulmonary artery, the alveoli and the lymph nodes, were stained with various stainings at the same site in order to give a comprehensive picture of their composition. Besides Haematoxylin&Eosin and Elastica van Gieson staining, collagen I, collagen III, fibronectin, α-SMA, ki-67 and protease-activated receptor-2 (PAR-2) immunohistochemistry was performed. Collagen I, collagen III and fibronectin expression was positive at the outer rim of the pulmonary arteries, while the inner rim was collagen III positive. Moreover, the fibronectin staining in the intrapulmonary bronchus showed an opposite trend when compared to the collagen III staining. The alveoli exhibited PAR-2 expression, while PAR-2 was not expressed in lymph nodes of the healthy rabbit lung.
Highlights
Pulmonary diseases, such as bronchopulmonary dysplasia, require pre-clinical animal models to be elucidated and studied in detail (Salaets et al, 2020)
We cover basic aspects of paraffin tissue sec tions fixed with formaldehyde compared to cryopreservation; an addi tional antigen retrieval (AR) step compared to immunohistochemical staining without Antigen retrieval (AR) in the case of paraffin sections, as well as chro mogenic versus fluorescence staining, respectively
Immunohistochemical staining for collagen I (Fig. 1) with DAB as chromogen revealed that at lower magnifications, morphologies of bronchi, arteries and veins had a higher contrast to the staining intensity of the surrounding tissues, such as alveoli, intra-alveolar septa, alveolar sacs and ducts, in the cryosections compared to the paraffin sections
Summary
Pulmonary diseases, such as bronchopulmonary dysplasia, require pre-clinical animal models to be elucidated and studied in detail (Salaets et al, 2020). Inflammation related markers and their protein expression in the lung are important tools to investigate devastating lung diseases such as COVID-19 (Helmy et al, 2020) In this technical note, we cover basic aspects of paraffin tissue sec tions fixed with formaldehyde compared to cryopreservation; an addi tional antigen retrieval (AR) step compared to immunohistochemical staining without AR in the case of paraffin sections, as well as chro mogenic versus fluorescence staining, respectively. We have assessed Haematoxylin&Eosin (HE) and Elastica van Gieson (EvG) staining in the healthy rabbit lung tissue, collagen I, collagen III, fibronectin, alpha smooth muscle actin (α-SMA) and protease-activated receptor-2 (PAR-2) staining, the latter being an inflammation related protein on the cell surface, potentially interesting in studies of lung injuries, pulmonary diseases, fibrosis (Richeldi et al, 2017) and COVID-19 (Shanmugaraj et al, 2020). The images provided for the rabbit lung here may be used for comparison to other animal species or to the human lung
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