Avidin biotin pk6100

Hematoxylin and Eosin (H&E) staining was performed in 6-μm thick sections of formalin-fixed paraffin-embedded (FFPE) tissues. For immunohistochemistry, FFPE sections were antigen retrieved using citrate buffer (10 mM sodium citrate and 0.05% tween-20 pH 6.0) at 95°C for 15 min. Next, peroxidase blocking was performed for 15 min using methanol containing 0.3% H₂O₂, followed by Avidin/Biotin (PK6100, Vector Labs) blocking for an additional 15 min at room temperature. The sections were then blocked with 5% goat serum in PBST for 30 min at room temperature and incubated overnight with a primary antibody against TOM20 (Invitrogen, Waltham, MA, USA) in a humidified chamber at 4°C. The sections were subsequently stained with a secondary antibody for 1 h at room temperature and developed using DAB Peroxidase Substrate kit (Vector Labs, Burlingame, CA, USA). Images were acquired using Olympus Provis microscope and processed for noise background equally.

Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 7 μm. Antigen retrieval was processed after rehydration in citrate acid buffer (pH 6.0) at 95°C for 15 min. Methanol containing 0.3% H₂O₂ was used for peroxidase blocking for 15 min, followed by Avidin/Biotin (PK6100, Vector Labs) blocking for additional 15 min at room temperature. The sections were then blocked with phosphate-buffered saline (PBS) containing 10% goat serum for 1 hour at room temperature and incubated overnight with the anti- UCP1 primary antibody (1:200; ab10983, Abcam) at 4°C. The sections were subsequently stained with secondary antibody for 1 hour at room temperature. Images were acquired with Leica DM2000 digital camera.