ICC Antibodies

Immunocytochemistry (ICC) is a microscopy-based technique that uses antibodies for detecting targets of interest in cells. It typically employs an immunofluorescence (IF) readout since this allows for multiple analytes to be visualized simultaneously. As well as staining samples with fluorophore-labeled antibody reagents, it is common for researchers to include fluorescent nuclear counterstains such as 4', 6-diamidino-2-phenylindole (DAPI), Hoechst 33342, or propidium iodide (PI) in experimental protocols to help put the resultant ICC data into context. The types of samples that are studied by ICC include adherent cell cultures that have been grown as monolayers on the surface of microplate wells, and suspension cells that have been applied to glass slides. ICC is also used for studying more complex three-dimensional (3D) structures like organoids and spheroids, although these kinds of experiments generally involve adapting the standard ICC protocol. For example, 3D cultures must often be released from the growth matrix prior to immunostaining and a clearing step may be required to prevent light scatter during imaging. When working with standard sample types, the ICC workflow begins with fixation to preserve the cells at a set point in time and prevent target degradation. This usually involves incubation in a dilute solution of paraformaldehyde (PFA) at room temperature, or in neat methanol at -20^oC, for 10 minutes. If a PFA fixative is used, and the aim of the ICC experiment is to detect intracellular analytes such as mitochondrial markers, the cells must next be permeabilized to allow antibodies to access their targets. However, permeabilization is unnecessary when working with methanol-fixed samples. Following fixation and permeabilization, the samples are blocked to prevent non-specific antibody binding. Bovine serum albumin (BSA) is widely used for blocking, although researchers may also choose to use gelatin or normal serum. Immunostaining can then proceed, either using fluorophore-labeled primary antibodies for direct detection, or a combination of unlabeled primary antibodies and labeled secondary antibodies for indirect detection. When normal serum is used for blocking prior to indirect detection, it is recommended that the serum shares the same host species as the secondary antibody reagent. A main advantage of direct detection is that it shortens the ICC workflow by eliminating the secondary antibody incubation step and a series of washes. It also allows for the use of several primary antibodies from the same host species, simplifying experimental design when performing multiplexed experiments. Indirect detection can provide signal amplification to increase the likelihood of detecting scarce targets, as well as offering greater flexibility for fluorophore selection due to the broad range of labeled secondary antibodies that are available. Once immunostaining is complete, including counterstaining, microplate-based samples are submerged in PBS and can proceed to imaging. Slide-based samples must instead be mounted to protect them from contact with the microscope lens and prevent them from drying out. Commercial mounting media are designed to offer various benefits, such as rapid setting, reduced photobleaching, and stable long-term storage. Explore our range of ICC antibodies below and discover more, for less.