Rodent decloaker buffer

4 μm paraffin section were mounted on charged glass slides (Superfrost® Plus, Thermo Scientific, Menzel-Gläser) and pretreated with Rodent decloaker buffer (BioCare) at pH 6 in a 2100 automated pressure cooker (PickCell). Consecutive sections were incubated overnight with a polyclonal guinea pig anti-mouse Perilipin2 (Progen, GP40) in 1:2000 dilution, and monoclonal rabbit anti-mouse SCD1 (Cell Signaling #2794) in dilution 1:100. For the Perilipin2 antibody a mouse on mouse detection kit (Vector, BMK-2202) was used according to the manufacturer’s protocol. Anti-Scd1 incubated sections were blocked in 4% normal goat serum and incubated with biotinylated goat anti rabbit (Dako, E0432) at a 1:300 dilution. Immunoreactivity was visualized using routine avidin-biotin amplification and diaminobenzidine (DAB) chromogenic reaction.

Sections (8 µm) were deparaffinized in xylene and hydrated in a series of graded alcohols to water, and then slides were immersed in a blocking reagent (Biocare’s Peroxidazed 1 blocking reagent, Biocare, Yorba Linda, CA, USA) for 5 min at room temperature (RT) and subsequently washed in distilled water. For heat-mediated antigen retrieval, the slides were placed in Rodent Decloaker buffer (Biocare, Yorba Linda, CA, USA) and heated to 100 °C for 30 min by a steamer; when the slides became cold, Rodent Block M (Biocare, Yorba Linda, CA, USA) was applied for 30 min. After washing with tris-buffered saline (TBS), the primary antibody (monoclonal anti bromodeoxyuridine, Biocare, Yorba Linda, CA, USA) was applied in a ratio of 1:100 (v/v) for 2 h at RT. This antibody reacts with BrdU in single-stranded DNA, for BrdU attached to a protein carrier or free BrdU. It detects nucleated cells in the S-phase that have had BrdU incorporated into their DNA. The samples were subsequently washed in TBS and then covered with Mouse-on-Mouse HRP-Polymer (Biocare, Yorba Linda, CA, USA) for 20 min at RT. After thoroughly washing in TBS, the slides were incubated for 5 min with a chromogen solution (Biocare’s DAB, Biocare, Yorba Linda, CA, USA). Positive and negative controls were performed as well.