#immunostaining

Immunostaining is the use of an antibody-based method to detect a specific protein in a sample. This post is an overview / contents post from which to link my other posts and try to put some order in this blog! 

Immunohistochemistry (IHC)

Immunostaining of cells of fixed or frozen cells (see my IHC post)

A primary antibody is added which sticks to the protein that’s being investigated.

A secondary antibody, linked to an enzyme, is added which then sticks to the primary antibody .

A chromogen is added, which the enzyme reacts with to deposit an insoluble coloured compound onto the cell.

This colour is detectable by light microscopy.

Therefore, the areas where that protein is present can be deduced by coloured areas visible down the microscope. 

Alternatively, radioactive elements can be used as labels, and the immunoreaction can be visualized by autoradiography.

Tissue is fixed to preserve cell morphology and tissue architecture (see my post on this process). 

Some antigens don’t survive formalin fixation. For samples needing these immuno tests, the tissue is rapidly fresh frozen in liquid nitrogen and cut with a cryostat before IHC.

The disadvantages of frozen sections include poor morphology, poor resolution at higher magnifications, difficulty in cutting over paraffin sections, and the need for frozen storage. 

Western blotting

(See my Western blot post) Western blotting allows the detection of specific proteins from extracts made from cells or tissues.

Proteins are separated by size using gel electrophoresis 

Smaller molecules move through the gel faster than larger molecules, and so travel further.

The separated proteins are transferred to a synthetic membrane via dry, semi-dry, or wet blotting methods. 

Antibodies, linked to something that will allow visualisation eg enzymes that produce a coloured product, are added to the membrane. 

Enzyme-linked immunosorbent assay (ELISA)

(See my ELISA post) The ELISA quantitatively or semi-quantitatively determines protein concentrations from blood plasma, serum or cell/tissue extracts in a multi-well plate format (usually 96-wells per plate). 

An antigen is immobilized on a solid surface

It is complexed with an antibody that is linked to an enzyme.

The conjugated enzyme activity is assessed via incubation with a substrate.

Which produces a product that can be measured.

Immuno-electron microscopy

Electron microscopy or EM studies detailed microarchitecture of tissues or cells.

 Immuno-EM allows the detection of specific proteins in ultrathin tissue sections. 

Antibodies labelled with heavy metal particles (e.g. gold) can be directly visualised using transmission electron microscopy.

Antigen retrieval on formaldehyde-fixed tissue sections is used to expose antigenic sites and allow antibodies to bind. Formaldehyde fixation results in protein cross-linking (methylene bridges), which masks epitopes and can restrict antigen-antibody binding. 

Heat-induced epitope retrieval

Heating with microwaves can result in uneven epitope retrieval due to hot and cold spots. 

Tissue can also be boiled off the slide.

Proteolytic-induced epitope retrieval

Uses enzymes such as proteinase K, trypsin, or pepsin to unmask antigen . 

Useful for epitopes that are difficult to retrieve.

Can sometimes damage the morphology of the section – concentration and timing need to be optimized.