Histological effects
Immunofluorescence (IF) staining was performed to evaluate the histological effects of protamine exposure (inflammation) and GAG-replenishment on urothelial barrier markers: chondroitin sulfate (CS), tight junctions and adherence junctions (Table 1). To induce mild-to-moderate inflammation, the cells were treated with protamine sulfate 1400 IE/mL for 4h at 37°C in 5% CO2 (LEO Pharma, Neu-Isenburg). [13] The cells were treated according to three different instillation protocols: 1) protamine sulfate (4h), 2) protamine sulfate (4h) & CS 0,2% (2mg/mL) (Gepan Instill, Pohl-Boskamp GmbH & Co., Hohenlockstedt) (1h) and 3) controls; instilled with Hank’s balanced salt solution (Thermofisher, catalogusno. 88284). Samples were then treated with 3% paraformaldehyde in PBS and air-dried. After this, samples were rinsed with 0.2% triton in PBS and incubated with primary and secondary antibodies for respectively 60 and 30 min. A list of antibodies is described in table 1. Slides were mounted using Dako mounting medium. Bright-field and epifluorescent binocular microscopy (Zeiss) and ImageJ (version 1.46) software were used for analysis.