Immunohistochemistry of isolated cells

Then SVF cells and ceiling cultures were fixed using 4% PFA for 10 min adipocytes encapsulated in Collagen I were fixed for 30 minutes. Cells were permeabilized with 0.2% triton-X washed and then blocked with 5% normal donkey serum, and 0.1% tween-20 overnight at 4C. Primary antibodies were then added in a 1% normal donkey serum and 0.1% triton x solution and incubated overnight at 4C. Secondaries including Phalloidin (F-actin) in SVF cells, BODIPY (lipids) in adipocytes, and DAPI (nuclei) were then added in a 1% normal donkey serum and 0.1% triton x solution. Cells were then washed and imaged using a Keyence BZ-X710 microscope (Keyence, Elmwood Park, NJ).