Anti rabbit hrp dab staining kit

Exploratory experiments were performed in four mice per group to qualitatively evaluate the content of smooth muscle within the airway wall. After collection, the left lung was perfused with 4% paraformaldehyde, fixed at 4°C for 3 days, embedded in paraffin, cut in 5 μm-thick sections and mounted on glass slides. Paraffin was then removed by 2 washes in Toluene for 5 min each. Then, tissue was rehydrated by successive baths in 100%, 95%, 70% and 50% of ethanol for 3 min and then washed in dH₂O. Antigen retrieval was performed by bathing slides in citrate buffer (pH 6) for 12 min at 95°C and then left bathing until the buffer reached room temperature. Slides were then washed in PBS and blocked for 1 h at room temperature in blocking buffer (1% BSA, 5% Normal Goat Serum, 0.3% Triton 100x in PBS). Slides were incubated overnight at 4°C with anti α -SMA (Abcam #ab5694) diluted 1:100 in PBS containing 1% BSA, 1% Normal Goat Serum, 0.3% Triton 100x. Staining was then visualized by using the anti-rabbit HRP-DAB staining kit from R&D system (CTS005) according to the manufacturer’s protocol. The images were converted into high-resolution digital data using the NanoZoomer Digital scanner (Hamamastu Photonics, Bridgewater, United States). Three to five lung sections per mouse were analyzed.