Question

1. What is the major difference between western blot and Immunohistochemistry? 2. What is the difference between a monoclonal and polyclonal antibody? 3. When is antigen retrieval necessary? What is the developing reagent used for immunohistochemistry in this weeks practice? How this reagent different from the substrates used in ELISpots and ELISA assays? 4.

Answer 1

1. The principle of detection is more or less the same. With Western Blot we separate proteins by size, and semiquantification is possible. The great advantage of IHC is that to see where in the cell protein is localized. This is of special interest if looking at proteins that change localization for example upon activation (eg. transfer from cytoplasm to nucleus, or from ER to cell membrane). If just want to show wheter a protein is present or not, Western Blot might be the method of choice.

2. Monoclonal antibodies are made using identical immune cells that are all clones of a specific parent cell. As such, they will have affinity for the same antigen and epitope (i.e. are monovalent). Polyclonal antibodies are made using several different immune cells. They will have affinity for the same antigen but different epitopes.

3. The need for antigen retrieval will depend a lot on specific antigen and the primary antibody we will use to immunolabel it. Some antigens are very robust or very abundant and can withstand strong fixation and still yield good immunolabeling. Antigen of interest may withstand PLP fixation just fine, but you may find it necessary to do antigen retrieval. However, we may also find that will need antigen retrieval to get any labeling at all, or that it will improve staining and make it more consistent.