The tissue sections were immersed in a 3%H2O22 solution for 30 min, to block endogenous peroxidase activity and submitted to an antigen retrieval heating step following the protocol recommended by the manufacturer (see Table 1).
Briefly, tissue sections were fixed, de-waxed and rehydrated prior to antigen retrieval (heating in a microwave at full power for 20 min followed by cooling to room temperature).
Sections were fixed for 3 min in 4% paraformaldehyde (PFA) (no PFA fixation for MyHC antibodies), and if needed, subjected to antigen retrieval: heating slides in citrate buffer (10 mM sodium citrate, pH 6.0) in a steamer (Oster 6.1 quart, model 5712, Racine, WI) for 15 min followed by cooling at room temperature for 2 min (Tang et al., 2007).
Before the second staining with anti-Foxp3, frozen tissues were subjected to Ag retrieval by heat treatment in citrate (pH 6) buffer.
Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1 50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).
For immunohistochemistry, 4-µm thick skin sections were deparaffinized and subjected to antigen retrieval by heating.
Following sectioning and antigen retrieval by heating in 10 mM sodium citrate pH 6.0, sections were washed in PBS and endogenous peroxidase was blocked in 3% (v/v) H2O2 in methanol.
After deparaffinization and rehydration, the tissue sections were subjected to antigen retrieval by heating in a microwave for 10 minutes in Vector H-3300 antigen unmasking solution (1∶100; Vector Laboratories, Inc., Burlingame, CA).
Tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval by heating in target antigen retrieval solution (DakoCytomation, Carpinteria, CA, USA) according to the manufacturer's protocol.
