Tissue preparation is crucial to any immunohistochemical procedure. The success of the application is based on antigen preservation and tissue morphology. These variables can be optimized if proper consideration is given to the fixative, method of fixation, tissue sample size, length of time in and temperature of the fixative, and whether the sample is frozen or embedded in paraffin blocks. The availability of primary antibodies that work well in mouse tissues is of primary concern. Some of the antibodies work better in frozen tissue, and some work better in paraffin-embedded tissues. Some antibodies are not commercially available. If specific antibodies are not available, individual investigators can be a good resource. One must consider tissue morphology when deciding on freezing versus paraffin embedding samples. It is faster to freeze the tissue, but the morphology is often compromised. It is time-consuming to embed samples, but the results are often enhanced due to improved morphology. There is not a universal fixative, so it is important or ideal to use a fixative that is known to work for the tissue type and target antigen. If IHC is to be performed on tissue that has been fixed in formalin, it is important to keep in mind that antigens may be masked. Antigen retrieval may be performed by steaming the slides in citrate buffer for 10 min. This method tends to work well and results in minimal destruction of cellular morphology. It is important to keep in mind that different antigen-antibody combinations may require different retrieval methods. Once the tissue is properly prepared, one can follow specific protocols for staining paraffin sections or frozen sections. The majority of the protocols involve blocking of various tissue components, antibody and detection reagent incubations, and buffer washes. For more detailed information, one should consult references discussing general techniques and specific applications.16

0 0

Post a comment