The Immuno part of histology, in general is definitely more difficult than traditional staining methods. The IHC protocols are a lot like the dyes protocols but also are not. IHC protocols are more complicated, take longer and reagents are much more expensive. Antibodies (primary and secondary) need to be kept sterile at all times or else they will not work the next time you go to use them.
Figuring out the protocol (Antibody optimization) can be a huge undertaking. Part of that antibody optimization is the decision to use Heat-Induced Epitope Retrieval (HIER) reagents. HIER reagents are generally used to unmask epitope sites on formalin fixed paraffin embedded tissues.
Frozen section epitopes can usually be unfolded using enzymes or acids. If your testing an antibody, try the enzyme reagents before you use any of the acid protocols. The most used enzymes and acid protocols, that we found useful can be downloaded here.
Most of these reagents we made ourselves, onsite, QC’d and tested. Is it easier to just buy them? It depends on your training and level of expertise. If you’ve never made them before, then you best bet is to buy them. Keep in mind, the cost to buy pre-made reagents is way beyond purchasing the individual salts and mixing it yourself.
Tris Buffer – This is a link to a website.