Hello everyone, I’m currently working with human osteosarcoma spheroids for histological analyses. After embedding the spheroids in agarose and infiltrating them, as well as embedding them in paraffin, I’ve noticed that the spheroids sections have a strange structure. They are almost destroyed and present no extracellular matrix (ECM). I’m using a protocol that was established many years ago in our lab, but something seems to be going wrong. I’ve tried to fix my spheroids with formaldehyde, both with and without methanol. I’ve also avoided centrifuging the spheroids in the embedding medium. I’ve tested two different embedding media, 2% agarose in PBS and 2% agarose dissolved in Histogel. However, I keep getting the same strange images. The H&E staining works properly with a control sample (mouse kidney section), so the issue is not due to the staining solutions or protocol. Although I’m not an expert, I’m wondering if the dehydration and infiltration steps could be the problem? Thanks a lot in advance for your support!