# Urokinase System in Pathogenesis of Pulmonary Fibrosis: A Hidden Threat of COVID-19
## 4 Materials and Methods
### Immunohistochemistryistochemistry, Microscopy and Morphometric Analysis  
Five \(\upmu\)m-thick formalin-fixed, paraffin-embedded sections of human parenchymal lung tissues were deparaffinized in xylene and rehydrated in a gradually decreasing concentration of methanol (100%, 95% and 70%); antigen retrieval was carried out with Trilogy buffer (Sigma-Aldrich, Merck Group, Darmstadt, Germany) according to the manufacturer's protocol. The slides were then rinsed 2 times with PBS for 5 min. Endogenous peroxidase was blocked using 3% hydrogen peroxide solution for 20 min; the slides were then washed 2 times with PBS for 5 min. Nonspecific binding was blocked with 1% BSA/PBS blocking solution for 30 min at room temperature. The slides were incubated with primary antibodies against uPA ([PERSON], San Diego, CA, USA cat #369002), uPAR (Abcam, Cambridge, UK, ab103791) or ACE2 (CloudClone, Wuhan, China, PB886 Mu01); diluted 1:100 in blocking solution for 1 h at room temperature; and washed 2 times with PBS for 5 min. Further, slides were incubated with secondary ready-to-use peroxidase-conjugated Real EnVision antibodies (Dako, Carpinteria, CA, USA) for 30 min at room temperature for single staining or with AP horse anti-mouse IgG (ImmPRESS HRP/ AP Polymer System, Vector laboratories, Burlingame, CA, USA) for double staining for 30 min at room temperature, followed by 3 washes with PBS for 5 min. In the case of single staining, HiDef Detection(tm) HRP Polymer Detector (HiDef Detection HRP Polymer system, Cell marque, Rocklin, CA, USA) was also applied for 10 min at room temperature, followed by a rinse with PBS for 5 min. For single staining, DAB chromogen substrate (Dako, Carpinteria, CA, USA) was added for 2 min, and the reaction was stopped in distilled water. For double staining, ImmPACT DAB EqV Substrate incubation was followed by ImmPACT Vector Red Substrate (ImmPRESS HRP/AP Polymer System, Vector laboratories, Burlingame, CA, USA) for 2 min, and the reaction was stopped in distilled water. Finally, the slides were dehydrated in graded methanol (70%, 95%, 100%) and xylene baths and mounted with Cytoseal(tm) 60 (Thermo Fisher Scientific, Waltham, MA, USA).  
The slides were scanned using Aperio ImageScope (v12.4.3.5008, Leica Microsystems GmbH, Wetzlar, Germany). Quality control of the scanned images and all further analysis were performed using Aperio ImageScope. All images were analyzed with the exclusion of internal contents of blood vessels using the negative pen tool to eliminate nonspecific detection in this area. Slides were analyzed using the Positive Pixel Count v9 algorithm, which counts pixels of a predetermined color, intensity and saturation. The algorithm input parameters were initially set to obtain the identification of pixels related to brown or pink (positive pixels) and the identification of pixels related to other colors (negative pixels). The algorithm output is composed of the number of positive pixels (Np) and the number of negative pixels (Nn). A staining score is then defined and calculated by the algorithm as Np/(Np+Nn).