Following antigen retrieval,В cooling to roomВ temperatureВ and washing,В tissues were blocked in 3% BSA (Product # 37525) in PBST for 30 min at room temperature and then probed with an Ezrin monoclonal antibody (Product # MA5-13862) at a dilution of 1:100 for 1 h in a humidified chamber. IHC was performed onВ aВ formalinвЂЇfixed,вЂЇparaffinвЂЇembeddedвЂЇ(FFPE)В human colon cancer tissue section.В To expose target proteins, heat-induced epitope retrievalВ (HIER)В was performed using 10В mM sodium citrateВ buffer, pH 6,В (e.g., 00-5000, AP-9003-125)В for 20 minВ by heatingВ at 95вЊ. Tissues that are too delicate for the rigorous processing involved with paraffin removal and antigen retrieval are first embedded in cryoprotective embedding medium, such as OCTВ compound, and then snap-frozen and stored in liquid nitrogen untilВ they areВ sectioned.В The example below provides an example of IHC staining in formalin fixed tissue. For example, blood smears areВ usuallyВ fixed by drying, which removes the liquidВ fromВ theВ sample and fixesВ the cellsВ to the slide. Physical fixationвЂЇis an alternate approach to prepare samples for staining, and the specific method depends on the sample source and the stability of the target antigen. No single fixative is ideal for all tissues, samples or antigens.В This means that each fixationВ procedureВ must beВ optimized toВ assureВ adequateВ fixationВ withoutВ alteringВ theВ antigen or disturbing the endogenous location and the cellular detail of the tissue. Type of fixative (formaldehyde, glutaraldehyde,В organic solvent,В etc.)Д¬hemical fixativesВ crosslink or precipitate sample proteins, which can mask target antigens or prevent antibody accessibility to the tissue target after prolonged fixation.

Therefore, the following considerations should be addressed when choosing a fixative: The guidelinesВ provided hereВ are helpful in determining the appropriate fixative for a particular system, butВ it is important to remember thatВ each antigen is unique. While a particular fixative may preserve the immunoreactivity of oneВ antigenicВ epitope, it may destroy others, even if they are on the same antigen.

Drying:вЂЇBlood smears for ICCВ stainingВ are air-dried and waved across a flame to heat-fix the cells to the slide.

Freezing:вЂЇSamples with antigens that are tooВ labileВ for chemical fixation or exposure to the organic solvents used for de-paraffinization can be embedded inВ aВ cryoprotective embedding medium, such asВ optimal cutting temperature (OCT)В compound, and then snap-frozen and stored in liquid nitrogen.

Immersion is often combined with perfusion to ensure thorough fixation throughout the tissue.

Immersion:вЂЇSamples are immersed in fixative which then diffuses into and through the tissue or cell sample.

Perfusion:вЂЇTissues can be perfused with fixative following exsanguination and saline perfusion to allow rapid fixation of entire organs.

This means that the optimal fixation method may have to be determined empirically.В Common methods of fixation include: The right fixation method requires optimization based on the application and the target antigen to be stained. It protectsвЂЇsamples against microbial contamination andВ possibleВ decomposition.It strengthensвЂЇsamplesВ so that they canВ withstand further processing and staining.It inactivatesВ proteolytic enzymes that could otherwise degrade the sample.It preserves andВ stabilizesвЂЇcell morphology and tissue architecture.Fixation plays four critical roles in immunohistochemistry:

Higher grade fixation software