60 Chapter 3 Immunohistochemical staining (IHC) IHC was used to quantify proteins of interest in lung tissue. Control lung tissues were obtained through resections from patients undergoing surgery for lung cancer, taken from as far away from the tumor region as possible and were macroscopically normal tissue as assessed by pathologists. Fibrotic lungs were obtained from explanted material following transplantations (Table 1). Formalin fixed and paraffin embedded tissue sections of 6 μm thickness were obtained from never-smoker (n=9), ex-smoker (n=9) and IPF (n=12) lungs and were stained for COL14A1 using polyclonal rabbit antiCOL14A1 antibody (HPA023781; Atlas antibodies Inc.). Secondary polyclonal Goat Anti-Rabbit antibody (P0488, Dako, Denmark) was visualized using NovaRED® Substrate (SK-4800, Vector Laboratories, Canada). Counterstaining was done using hematoxylin. Slides were scanned using a Hamamatsu NanoZoomer digital scanner (Hamamatsu Photonic K.K., Shizuoka, Japan) as described previously [10]. The study protocol was consistent with the Research Code of the University Medical Center Groningen (research code UMCG (umcgresearch.org) and national ethical and professional guidelines (“Code of conduct for Health Research (only in Dutch): Gedragscode-Gezondheidsonderzoek-2022.pdf (coreon.org). Lung tissues used in this study were derived from leftover lung material after lung surgery and transplant procedures. All samples and clinical information were coded before experiments were performed. This material was not subject to the Medical Research Human Subjects Act in the Netherlands; all biological samples are from archival materials that are exempt from consent in compliance with applicable laws and regulations (Dutch laws: Medical Treatment Agreement Act (WGBO) art 458 / GDPR art 9/ UAVG art 24) . Table 1: Patient characteristics for the donors included in this study. Never smoker Exsmoker IPF P value Never smoker vs IPF Ex-smoker vs IPF Age (median (min-max)) 70 (50-82) 55 (43-62) 58.50 (37-67) 0.0213* ns* Sex (M/F) 1/8 3/6 9/3 0.075# ns# FEV1 (pred) † (%, (min-max)) 107.5 (80-121) 103.6 (85-127) 49.35 (18-74.50) 0.0002 * 0.0002* *: Tested using Mann-Whitney test, #: Tested using Fisher’s exact test. †: Data was available only n=8 for all groups. IPF: Idiopathic pulmonary fibrosis, FEV1: Forced expiratory volume in 1 second. ns: not significant
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