Simple
CLEM
method to asses rare pulmonary vascular remodeling
Authors: Longin‐Péchoux Christine , Antigny Fabrice , Ranchoux Benoit , Gouadon Elodie , Humbert Marc , Perros Frédéric , Rücker‐Martin Catherine 
Published: December 2016

Simple CLEM method to asses rare pulmonary vascular remodeling

Péchoux C
1
; Antigny F
2
; Hautefort A
2
; Ranchoux B
2
; Gouadon E
2
; Humbert M
2
; Perros F
2
; Rücker‐Martin C
2

1‐ GABI, Inra, AgroparisTech, Université Paris‐Saclay, MIMA2, 78350 Jouy‐en‐Josas, France

2‐ Centre Chirurgical Marie Lannelongue, Inserm UMR‐S999, Le Plessis‐Robinson, France

Key Words: CLEM, PAH, FNG,

Pulmonary Arterial Hypertension (PAH) is a rare disease featured by obstructive lesions of small pulmonary arteries (Ø<500µm), such lesions representing only 1% of the pulmonary tissue. Recently, we evidenced the implication of the endothelial‐mesenchymal transition (EndoMT), the increase of phosphorylated vimentin (P‐Vim) and the decrease of KCNK3 channel in cellular processes at the origin of the occlusive vascular remodeling in PAH. For these purposes, we adapted the CLEM methodology usually performed on small samples to large tissue taking into account two major biological specificities. First, the lung is a soft organ, consisting of air‐filled alveoli, on which CLEM classical preservation methods cannot be applied because of the formation of large crystals which tear and disrupt structures. Second, PAH impacted arteries are 200‐500 µm large and represent only 1% of the total lung mass (Figure 1). Therefore, we have developed a complete fixation process based on constraints imposed by electron microscopy, suitable for immunoflurorescent labeling and an immunolabeling process responding to fluorescent confocal microscopy constraints (low autofluorescence) suitable for transmitted electron microscopy (pulmonary ultra‐structure preservation and use of various antibodies).

Fresh lung tissue was gently inflated through the bronchi using an automated formalin‐pump and immersed in buffered formalin for 24 hours. Then, lung samples (size 2×2.5 cm) were impregnated in increasing sucrose baths (10%/ 40%/ 40) before embedded in Tissu Tek® and frozen. Before immunolabeling, thick cryosections (10µm) were collected on gridded correlative slides, quenched with NH4Cl and nonspecific sites blocked. Sections were incubated with primary antibody before FluoroNanogold™ Conjugates. Pulmonary arteries of interest were identified under a LSM 700 confocal microscope. After confocal acquisition, sections were post‐fixed with 1% glutaraldehyde, quenched with glycine, incubated with the Gold reagent and fixed in osmium tetroxide. After dehydratation in ethanol, sections were embedded in Epon. Areas of interest identified by confocal microscopy were sectioned to perform TEM. Ultrathin sections (70 ‐ 80 nm) were collected onto 150 mesh cooper grids and counterstained with Oolong Tea extract (OTE) 0.05% and lead citrate before examination with a Hitachi HT7700 operated at 80kV.

In contrast to cryopreservation, this lung adapted methodology allows to preserve the tissue structure closed to results obtained with Epon embedding (Figure 2) and to follow PAH lesions unambiguously during all the steps. Moreover, such tissue fixation and embedding allow the antigens preservation without producing non‐specific labeling. In PAH lesions, confocal microscopy analysis was not resolutive enough to specifically identify, localize and quantify P‐Vim or KCNK3 labeling. CLEM approach confirmed Western‐blot results with significant increase in P‐Vim and decrease in KCNK3 in PAH lesions (Figure 3).

Hence, using correlative microscopy, we overcame the difficulties associated to the lung ultrastructure which is difficult to preserve during EM techniques (hydrophilic resins, Cryo‐TEM) and to the location and identification of the small arteries of interest. We found out a good compromise that allowed us to evidence the presence and the exact location of proteins in rare pulmonary lesions, little or not visible in confocal fluorescence microscopy.