Progres c10plus camera

To evaluate the predisposition for a Th1/Th2 tumor microenvironment prior to BCG, the density of Th1 and Th2 cells in tumor-infiltrating immune cells was measured on formalin-fixed, paraffin-embedded tissue sections of bladder cancer (in the lamina propria without invasion, at the invasive front, within the neoplastic urothelium and within the papillary stroma) by immunohistochemistry, using a T-bet antibody (monoclonal rabbit antihuman T-bet, MRQ-46, prediluted, Roche) and a GATA3 antibody (monoclonal mouse antihuman GATA3, L50-823, prediluted, Roche), which we had already validated in a recently work [30 (link)]. IHC staining was performed using an automated immunostainer (BenchMark ULTRA, Ventana Medical Systems, Tucson, US) according to the manufacturer’s protocol. We manually counted the total density of positive cells for each subset in up to 5 high-power fields (HPF) in each region, using the same field of view in consecutive slides. Microscope images were taken with an Olympus BX50 microscope (40x magnification) equipped with the ProgResC10plus camera (Jenoptik, Jena, Germany). IHC evaluation was performed by an experienced uropathologist.

The samples were fixed with paraformaldehyde (PFA). Briefly, 1 mL taken from the respective timepoint was centrifuged at 13,000 × g for 3 min to remove the supernatant and resuspended in 400 mL of PBS and 1.1 mL of PFA solution (4% in PBS, pH 6.9) at 4  °C overnight. Then, the tubes were centrifuged at 13,000 × g for 3 min to remove the supernatant, and a further three washes were performed with 1 mL PBS. Finally, the pellet was resuspended in 600 µL of ethanol (50% v/v) in PBS and stored at −20 °C. Optical microscopy was performed through an Olympus, Japan, BX60 optical microscope; micrographs were acquired with a Jenoptik ProgRes C10plus camera (Jenoptik AG, Germany). After fixation, the sample pellet was analysed in epifluorescence mode, and Calcofluor white dye was used in proportion 2:1 (dye:samples).

The following microscopes were used for monitoring toxin-induced cell rounding: Axiovert 40 CFL microscope (Carl Zeiss Microscopy, Jena, Germany) equipped with a ProgRes C10 plus camera (Jenoptik, Jena, Germany); Leica DMi1 equipped with a Leica MC170 HD camera (Leica, Wetzlar, Germany); Primovert (Carl Zeiss Microscopy, Jena, Germany); Lionheart FX Automated Microscope (BioTek, Vermont, United States). Toxin-induced cell rounding was manually quantified in microscopic images and, optionally, facilitated by the Neuralab online tool (https://neuralab.de).