I am planning an IHC stain using a polyclonal antibody that targets the phosphorylated version of the protein of interest. I know there are special considerations when doing western blot for phospho proteins (BSA as block rather than milk, special phosphatase inhibitors, etc) but is there anything I should keep in mind for IHC? Any considerations for polyclonal antibodies in general?
Hello Ajinkya R. Limkar
Special considerations while doing IHC for phosphoproteins are as follows:
1. The cold ischemia time i.e., the time a tissue specimen sits ex vivo prior to fixation, is a preanalytical variable that can have a profound effect on measurements of signaling proteins like phosphoproteins. A rapid placement of tissue into cold formalin fixatives will help to prevent some of the negative effects of prolonged cold ischemia time on measured levels of phosphoproteins.
2. Another factor which you should consider is the fixative. While formalin is routinely used as a fixative for paraffin embedded tissue samples, research has suggested that other fixatives may be better for the preservation of phosphoproteins. One such fixative is Streck’s Tissue Fixative (STF) which may be superior in preserving phosphoproteins compared with 10 % NBF or 4 % paraformaldehyde. Other alternative to formalin include a one-step room temperature process using biomarker and histology preservative (BHP). You may refer to the articles attached below for more information on alternative fixatives.
Article Choice of Fixative Is Crucial to Successful Immunohistochemi...
Article One-Step Preservation of Phosphoproteins and Tissue Morpholo...
Article The PAXgene® Tissue System Preserves Phosphoproteins in Huma...
3. You should use kinase and phosphatase inhibitors as they help to stabilize the activation status of proteins, preventing phosphorylation and dephosphorylation, respectively. Sample preparation techniques may involve kinase and phosphatase inhibitors at different stages depending on the process and purpose. For example, they may be included with the fixative or incubated with the sample prior to freezing. In fact, you should use a combination of kinase and phosphatase inhibitors which may stabilize proteins in a better way than in the presence of phosphatase inhibitor alone. It will help in both ways i.e., kinase inhibitors will block the upsurge in phosphorylation of phosphoproteins while phosphatase inhibitors will prevent dephosphorylation. See the paper attached below for more details.
Article A Portrait of Tissue Phosphoprotein Stability in the Clinica...
I am attaching a few more references below that may be helpful.
https://www.indivumed.com/fileadmin/documents/pdf/David-Juhl-2015-Article-Handbook-of-Practical-Immunohistochemistry-Chapter-8-2nd-edition-final.pdf
https://www.nature.com/articles/s41598-020-58257-3
Best.
One technical recommandation: don't use PBS because Phosphate buffer could lead to a negative result. Instead of this use TBS.
Nothing to add to Malcolm's detailed answer (other than to second the use of cold fixative until fixation is complete - keeping in mind that cold NBF tends to have slower tissue penetration, so keep sample size small/thin, and prolong the fixation as needed. On your polyclonal question: there is no problem using a polyclonal for IHC, as long as you've validated its specificity properly. For phosphorylated states that can be a bit tricky, depends on what you are trying to detect (phosphorylation at one site vs another site ? phosphorylated protein vs total protein ?), you need to have tested it on proper controls first, to be sure of your conclusions.
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