Hi everyone. We want to do an IHC in peripheral blood smear but it's our first time, and we have some doubts.
How do we have to fix the samples? Which is the best fixation agent, acetone, methanol or pfa? for how many time? Can we add the primary antibody directly after the fixation?
We need some tips.
The choice of the fixative depends on the used chromogen. In case of DAB, the better fixative is a mixture of ethanol (9 parts) and formalin (1 part). Timing: 10 seconds. Wash in tap water gently and air dry.
In case of alkaline phosphatase, you can use methanol-formalin (9+1)
Hello Raquel
I would pay close attention to the type of slides that you are using to make sure that your cells stay attached to it during the entire IHC protocol. You can either coat your slides with L-polylysine or use frosted charged slides. For the fixation PFA or acetone should work, I would pick depending on the optimal fixative for your antibodies.
I think (and I'm sure, for practicing it) that doing an IHC on a blood smear is not a good idea. Because the red blood cell is full of endogenous peroxidases and other parasitic enzymes that are very difficult to remove with H2O2 for example. The IFI would be more judicious. For fixation, the use of cold acetone on cells stuck by centrifugation is very useful because it allows the use of all antibodies capable of labeling a cell surface, intracytoplasmic or intranuclear. To label lymphocytes in IHC it is therefore preferable to work on purified lymphocytes on ficoll gradient. But beware of the residual GRs;
IHC is immunohistochemistry, the term that is applied on tissues. The correct trem for blood smear is cytochemistry. Yes, fixative depends on what marker you want to do. Mostly, cell markers are done by FACS.
I'm sorry but I disagree with the peremptory distinction provided by Lavender. Although the term "-Histo-" may suggest that the immunohistochemistry is only reserved for tissues, this method can also be used on peripheral blood smears or bone marrow or other cytologic materials. Conversely, the cytochemistry can be used not only on cytologic materials but on tissues even.
The main differences between these methodologies are:
- cytochemistry is a technique for identifying different cell types by using the search of chemical compounds inside the cellular cytoplasm (for example, some lipids or lipoproteins in the Sudan Black stain or mucopolysaccharides in the PAS reaction). However, if cellular enzymes are sought such as alkaline or acid phosphatase; peroxidase; some esterases, the method can be also called " cytoenzymatic"
- IHC techniques are based on antigen-antibody reactions on the cellular surface or in the cytoplasm for identifying cell types or other molecules on the surface. The antigen-antibody bond must subsequently be revealed by detection systems (e.g. Avidin-streptoavidine; secondary antibody conjugated enzymes).
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