Tris hcl edta ph 9.0 buffer

Tissue samples of different MPM histotypes were fixed in 10% buffered formalin and paraffin embedded. Sections of 4 µm in thickness were fixed with xylene, 100% EtOH, and 95% EtOH and then microwaved three times in Tris–HCl/EDTA pH 9.0 buffer (Dako, Milan, Italy) for 5 min and washed in Tris-buffered saline. After neutralization of the endogenous peroxidase with H₂O₂ (hydrogen peroxide) for 10 min, the sections were first incubated with PBS + 2% w/v BSA + 0.4% w/v Casein for 5 min in order to block the non-specific sites, and then probed with rabbit anti-human C1q (1:500) overnight at 4°C. The bound antibodies were revealed using the Vectastain Elite ABC horseradish peroxidase (HRP) kit (Vector Laboratories, DBA, Italy). Secondary antibodies were detected by 3-amino-9-ethylcarbazole (AEC) + high sensitivity Chromogen (Dako). The sections were counterstained with hematoxylin (Dako). Slides were examined under a Leica DM 3000 optical microscope and images were collected using a Leica DFC320 digital camera (Leica Microsystems, Wetzlar, Germany).

Tissues were fixed in 10% neutral‐buffered formalin, processed, and embedded in paraffin. Five‐micrometer‐thick sections were deparaffinized in xylene and rehydrated to water prior to microwave antigen retrieval in Tris/HCl/EDTA pH 9.0 buffer (Dako Cytomation, Glostrup, Denmark) and PBS washing. After neutralization of the endogenous peroxidase with 3% H₂O₂ for 10 min, the sections were incubated with protein blocking buffer for 10 min before undergoing incubation with the primary antibody. Anti‐Patched (Santa Cruz Biotechnology) staining was developed using DAB (Vector Laboratories, Burlingame, CA, USA) followed by Hematoxylin counterstaining (MilliporeSigma). H&E staining was performed on 5‐μm paraffin sections according to common methods.