3 amino 9 ethylcarbazole aec substrate

The 3T3-L1 fibroblasts were differentiated into mature adipocytes, as described above, and shock-frozen by ice-cold acetone (ROTH, Karlsruhe, Germany) for immunocytochemistry. The air-dried cells were incubated in PBS for rehydration. The endogenous peroxidase activity was blocked with 3% H₂O₂ (ROTH, Karlsruhe, Germany). To avoid non-specific protein binding, the cells were incubated in 10% bovine serum albumin (BSA, ROTH, Karlsruhe, Germany), 10% fetal calf serum (FCS, Sigma-Aldrich, Steinheim, Germany) and 10% chicken serum (Sigma-Aldrich, Steinheim, Germany), followed by 3 h incubation in a moist chamber with a polyclonal anti-TLR7 antibody from rabbit (2 µg/mL in 1% BSA; Invitrogen, Carlsbad, CA, USA). The cells were then stained with peroxidase-conjugated goat anti-rabbit IgG (1.6 µg/mL in 1% BSA; Jackson Immuno Research, West Grove, Pennsylvania, USA) for 90 min. The color development with 3-amino-9-ethylcarbazole (AEC) substrate (Vector Laboratories, Burlingame, CA, USA) at room temperature was stopped after microscopic examination. The rabbit isotype-matched IgG sera (ab37415; Abcam, Cambridge, UK) served as an isotype control. Parallel experiments without primary antibodies were carried out as negative controls.

The murine and human adipose tissue samples were fixed in ROTI^®Histofix 4% (Carl Roth, Karlsruhe, Germany) for 24 h and later embedded in paraffin. The paraffin was removed with xylene (ROTH, Karlsruhe, Germany) and by consecutive washing steps with 100%, 96% and 70% ethanol (ROTH, Karlsruhe, Germany). The endogenous peroxidase activity was blocked with 3% H₂O₂. Tissue samples were incubated for 60 min in citrate buffer at 60 °C. Non-specific binding sites were blocked with 5% BSA for 60 min in a humid chamber followed by an overnight incubation in a moist chamber with a polyclonal anti-TLR7 antibody from rabbit (2 µg/mL in 1% BSA; Invitrogen, Carlsbad California, USA). The cells were then stained with peroxidase-conjugated goat anti-rabbit IgG (1.6 µg/mL in 1% BSA; Jackson Immuno Research, West Grove, Pennsylvania, USA) for 90 min. The color development with 3-amino-9-ethylcarbazole (AEC) substrate (Vector Laboratories, Burlingame, California) at room temperature was stopped after microscopic examination. The rabbit isotype-matched IgG sera (ab37415; Abcam, Cambridge, UK) served as an isotype control. Parallel experiments without primary antibodies were carried out as negative controls.