Mouse smooth muscle actin clone 1a4

Preparations of brain, lung, heart, liver, kidney and gut from 8-12 weeks old C57BL/6J mice were frozen in −50°C 2-methylbutane, cut on a cryostat in 20 μm sections and mounted on glass slides. For tissue reactivity screening according to established protocols (Kreye et al., 2016 (link)), thawed unfixed tissue slices were rinsed with PBS then blocked with blocking solution (PBS supplemented with 2% Bovine Serum Albumin (Roth) and 5% Normal Goat Serum (Abcam)) for 1 hour at room temperature before incubation of mAbs at 5 μg/ml overnight at 4°C. After three PBS washing steps, goat anti-human IgG-Alexa Fluor 488 (Dianova, 109-545-003) diluted in blocking solution was applied for 2 hours at room temperature before additional three washes and mounting using DAPI-containing Fluoroshield. Staining was examined under an inverted fluorescence microscope (Olympus CKX41, Leica DMI6000) or confocal device (Leica TCS SL). For co-staining, tissue was processed as above, but sections were fixed with 4% PFA in PBS for 10 minutes at room temperature before blocking. For co-staining, the following antibodies were used: mouse Smooth Muscle Actin (clone 1A4, Agilent, 172 003), goat anti-mouse IgG-Alexa Fluor 594 (Dianova, 115-585-003). For nuclei staining DRAQ5 (abcam, ab108410) was used.