Hello Ibrahim. It depends on the antibody you are using, but normally, if you are using an adequate antigen retrieval treatment, you will not have any problems. Usually it is better to overfix than to underfix. Best of luck!!

Agree with Amadeu. usage of old fixed tissues for IHC is common . If the tissue stored in proper condition like ideal temp, it will be ok.

Thanks Amadeu for your answer but what do you mean by overfix than under fix ? It is the first time for me to run IHC ..... The sample still as organs in formalin Jars and I will fix them next week on slides (blocks) ... but still in jars from 3 months ago....

Thanks Reza for your answer ... the jars are stored in normal room temp ... around (20-25 c).

Immunohistochemistry is not an easy to do technology. Please consider some team work or intership with an expert before start doing it... :)

Like I wrote before working with overfixed samples depends on your antibody (and on your antigen retrieval and on your detection system...)

Underfixation means that your fixation was not enough to stablish the apropriate methylene bridges (crosslinks) between aa to preserve the tissues/cells/proteins/molecules.

Overfixation means that your fixation has established more than enough methylene bridges between aa to preserve the tissues/cells/proteins/molecules.

However, some say that because aa that are able to establish methylene bridges are not infinite in tissues, we can preview that after a number of methylene bridges being established, the fixative will stop its chemical fixation... So, they say, there is no infinite fixation, and, with appropriate antigen retrieval there is no problem working with overfixation.

There is some consensus that for IHC and for a sample of 10x10x3mm under fixation means less than 12 hours fixation, and overfixation means more than 72 hours fixation in 10% NBF at room temperature.

Some articles on the subject:

1 - Webster JD, Miller MA, Dusold D, Ramos-Vara J. Effects of prolonged formalin fixation on diagnostic immunohistochemistry in domestic animals. J. Histochem. Cytochem. 2009;57(8):753–761. doi:10.1369/jhc.2009.953877.

2 - Eltoum I, Fredenburgh J, Grizzle WE. Advanced Concepts in Fixation: 1. Effects of Fixation on Immunohistochemistry, Reversibility of Fixation and Recovery of Proteins, Nucleic Acids, and other Molecules from Fixed and Processed Tissues. 2. Developmental Methods of Fixation. J Histotechnol. 2001;24(3):201–210. doi:10.1179/014788801794812462.

3 - Mostegl MM, Richter B, Dinhopl N, Weissenböck H. Influence of prolonged formalin fixation of tissue samples on the sensitivity of chromogenic in situ hybridization. J. Vet. Diagn. Invest. 2011;23(6):1212–1216. doi:10.1177/1040638711425584.

4 - Sukhorukova EG, Zakhriapin MS, Anichkov NM, Korzhevskiĭ DE. [Microglia detection in the brain preparations after long-term storage in formalin]. Morfologiia. 2012;142(5):68–71.

5 - Webster JD, Miller MA, DuSold D, Ramos-Vara J. Effects of prolonged formalin fixation on the immunohistochemical detection of infectious agents in formalin-fixed, paraffin-embedded tissues. Vet. Pathol. 2010;47(3):529–535. doi:10.1177/0300985809359607.

That is Ok ... Thanks Amadeu

Is it possible to keep tissue specimen in formalin 10% for about 1 year and then do the ICH?  I want to collect the samples and when sampling is finished, then do the ICH.