Hi, I'm new here, and I have an interesting problem that I'm hoping someone can shed light on! Basically, I'm growing human coronary artery cells for IF (using labtek II chamber slides). I'm using gp340 as a primary and alexa 594 as secondary. After trying various fixatives, acetone is the best option for me(as pfa requires pre treatment and is too harsh on the cells). The other day I froze a slide in the -80, and did my IF the following week. The result was really positive in the frozen slide, but not in the fresh fixed. Does anyone know anything about the possibility of freezing making cells more permeable? Thank you!