Achieving uniform labeling of 3D cell culture models can be challenging and we see this as one of the main areas in which researchers struggle to adopt 3D cell culture models into their research workflow. For this reason, we have developed validated protocols for the labeling of 3D cell culture models that include detailed instructions and kits with everything you need to get started. Below we have several kits for our OpenLiver™ HepaRG™ NP 3D cell culture models:
Below is a general labeling protocol that can be used with these models for the labels described below.
- Following culture (and) treatment, 3D cell culture models are washed with PBS, labeled with a viability indicator at a dilution of 1:1000 in PBS for 30 min at room temperature, washed twice more, and fixed for 30 minutes in neutral buffered formalin at room temperature.
- After fixation, cells are washed, permeabilized with 2% Triton-X in PBS for 30 min, blocked with Visikol® HISTO™ Blocking Buffer for 1h, and then labeled with primary antibodies for 1h, at room temperature.
- For the primary antibody solution, antibodies against albumin, MDR1, IL8, and α-SMA can be used at a 1:200 dilution (1:100 for half-volume exchanges).
- Following incubation with primary antibodies, 3D cell culture models are washed twice with Visikol HISTO Washing Buffer, once with PBS, once with deionized water, and then the models are dehydrated through a methanol gradient from 50% to 100% and ultimately placed into Visikol® HISTO-M™ for imaging.