Immunochemistry

[sherly]

Hai,
I am working on aromatase gene and from my RT PCR data i have seen that there is a very significant difference in the expression of the gene with age. can i use immunohistochemistry to study the protein expression and how can i quantify the expression? Since the gene expression is very different, can i expect similar significant difference in the staining pattern too? thanks in advance.
Regards,
Sherly.

[danfive]

To quantify you need reliable standards....Then you can use ELISA, WB, ISH.

[sherly]

Thankyou for the reply. but still can immunohistochemistry be used to study the increase in staining and thereby indicating an increase in the expression? or is it just to locate the expression site? i donot have sufficient quantity of the antibody to do WB. I am trying ISH, but isn't it also used as IHC to locate the expression site? if ISH can work, why is it that IHC cannot?

[inmuno]

IHC is mainly a cualitative method, but you can also quantify if you have different groups to compare (number of cells, areas or even diferent intensity in some cases (ER or PR cuantifications in breast carcinoma for example)). IHC is very usefull to confirm the results of techniques like PCR or WB (ausence or presence of the protein in the tissue and where is located)
Other usefull technique could be Flow Cytometry.
I would stain a small number of samples first just to see what happens...
hope this could help you....
Best regards

[sherly]

Thank you for the information.

[radam2k]

You can also try quantitative image analysis, or even manual quantitation, to evaluate the expression. Of course, if you are attempting to assess a gradient of expression, you need to make sure that you have the antibody titred correctly to show the difference in expression.