Goat normal serum

The FFPE tissue blocks of the cases with target-sized amplicons were sliced at a width of 4 μm and placed on Immuno Coat Slides (Muto Pure Chemicals, Tokyo, Japan). The tissue slides were deparaffinized with xylene and rehydrated in a gradient series of ethanol solutions using a standard protocol. Antigens were retrieved by boiling in 5% Trilogy (Cell Marque, Rocklin, CA, USA) using Montage Opus^TM (Diagnostic Biosystems, Pleasanton, CA, USA) for 20 min. Thereafter, the tissues were blocked with 5% goat normal serum (Dako, Glostrup, Denmark) at room temperature (RT) for 1 h and then interacted with a monoclonal Chlamydia trachomatis antibody (Fitzgerald Industries, Concord, MA, USA) (showing cross-reactivity with C. psittaci) diluted 200-fold in Primary Antibody Diluent (Tris Green; ScyTek Laboratories, Logan, UT, USA), at RT for 1 h. Endogenous peroxidase was inactivated by 1% hydrogen peroxide for 10 min at RT, and following three washes with TBST, BioGenex^® Super Enhancer ™ (BioGenex, San Ramon, CA, USA) was applied at RT for 30 min. Development was performed using Dako EnVision+ System-HRP 3,3-diaminobenzidine (Dako) with hematoxylin counterstaining. The brown spots within the susceptible cells were interpreted as a positive signal indicating the presence of Chlamydia.

Tissues were embedded in O.C.T. compound (VWR) and 5-μm-thick fresh cryosections on positively charged microscope slides (Superfrost/Plus; Thermo Scientific) were fixed with 4% paraformaldehyde or acetone-methanol (1:1 v/v). For H&E or immunohistochemistry, sections were blocked and permeabilized (0.2% Triton X-100, 5% goat normal serum (DAKO) or 1% BSA (Sigma), stained (anti-neutrophil antibody [NIMP-R14] (ab2557, Abcam), polyclonal E. coli antibody (1:100, NB200-579, Novus Biologicals), anti-IL-1 beta (1:50, ab9722, Abcam) or anti-MMP-7 (1:100, ab4044, Abcam), all rabbit antibodies). Alexa 488 anti-rat IgG or anti-rabbit IgG and Alexa 568 anti-rabbit IgG (A-21210, A-11001 and A-11011, Life Technologies) were secondary antibodies and nuclei were counterstained with DAPI (0.05 mM, Sigma-Aldrich). Imaging was by fluorescence microscopy (AX60, Olympus Optical). Richard-Allan Scientific Signature Series Hematoxylin 7211 and Eosin-Y 7111 (Thermo Scientific) were used to counterstain the tissue sections.
Histology was scored using H&E stained bladder sections. The score was based on neutrophil infiltration, tissue architecture and epithelial thickness on a scale of 0–10, where 0 is unchanged compared to uninfected controls and 10 the highest neutrophil infiltration, most destroyed tissue architecture and maximum epithelial thickness.