ABSTRACT
The mandatory usage of extracellular matrix (ECM) gels in 3D cultures limits antibody penetration and increases background, while the removal of ECM gel causes disruption of morphology and sample loss. These factors pose challenges to effective immune labeling-based staining. Here, we present a protocol for whole-mount immunofluorescence staining of gel-embedded pancreatic organoids. We describe steps for sample fixation, blocking, and antibody incubation. We detail procedures for washing antibodies and mounting.
Subject(s)
Extracellular Matrix , Fluorescent Antibody Technique , Organoids , Pancreas , Organoids/cytology , Organoids/metabolism , Extracellular Matrix/metabolism , Extracellular Matrix/chemistry , Pancreas/cytology , Pancreas/metabolism , Fluorescent Antibody Technique/methods , Animals , Staining and Labeling/methods , Humans , Gels/chemistry , MiceABSTRACT
Neoadjuvant therapy with conventional chemotherapies have visibly improved the prognosis of locally advanced pancreatic cancer (PCa). However, molecular targeted therapies that have provided durable responses in other tumor entities, have not yet found access into neoadjuvant therapy of PCa. In fact, due to the presence of the tumor burden serving as an antigen source for T cell priming, neoadjuvant chemotherapy may unleash a more potent antitumoral immune response than adjuvant or palliative chemotherapy.