The antigen of interest I am going to detect (Paraffin embedded) is expected to be low in abundance. On the top of that my experiment demands use of mouse liver tissue fixed in 4% PFA for more than 2 months.
Whether that much over fixation affect antigenicity? Do I need to treat sections normally or need special procedures for antigen retrieval? Or the antigens would be completely masked irreversibly and may be devoid of antigens (as the case may be)?
How would you address this problem, any opinion?
Just only pointing to eventually interesting literature (apologize for lengthiness!).
It seems there IS an effect of "overfixation" using 4% PFA...and some special literature can be found also especially for IHC/antigens. 2 months fixation might be a bit too long...
At least some thoughts and ideas/results on degradation of DNA after fixation with paraffin (normal duration and circumstances and "overfixed/prolonged fixation time, etc...)
[DNA degradation in formalin fixed tissues] GERMAN original TITLE: 'DNA-Degradation in formalinfixierten Geweben'
Peter Wiegand, J. Domhöver, Bernd Brinkmann
link.springer.com/article/10.1007/s002920050185 = http://link.springer.com/article/10.1007/s002920050185 Der Pathologe, December 1996, Volume 17, Issue 6, pp 451–454
Abstract
The intensity of DNA degradation in fixed tissues is dependent on the fixation solution and the fixation time.
The aim of this study was the investigation of DNA degradation over fixation times of up to 70 days in different tissues (muscle, brain, liver, bone) and with different formalin concentration (2%, 4%, 8%; unbuffered).
An additional test was performed to see whether the fixed tissues could be individualized using PCR analysis.
The smallest amounts of DNA were extracted from liver and brain and the largest from muscle and bone.
The amount of DNA that could be extracted decreased with increasing formalin concentration, while at the same time DNA degradation increased.
With the PCR-VNTR system HUMTH01, all fixed samples could be typed regardless of the fixation time and the formalin concentration.
Similar articles
Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: an analysis of resected gastric carcinoma.[Pathol Int. 1996] https://www.ncbi.nlm.nih.gov/pubmed/9110353
Effect of buffered formalin on amplification of DNA from paraffin wax embedded small biopsies using real-time PCR. [J Clin Pathol. 2004] https://www.ncbi.nlm.nih.gov/pubmed/15166276
The effect of formalin fixation on restriction endonuclease digestion of DNA and PCR amplification. [Pathol Res Pract. 1993] https://www.ncbi.nlm.nih.gov/pubmed/8378178
Evaluation of DNA extraction methods and real time PCR optimization on formalin-fixed paraffin-embedded tissues. [ Dedhia P, Tarale S, Dhongde G, Khadapkar R, Das B. Asian Pac J Cancer Prev. 2007 Jan-Mar; 8(1):55-9. ] https://www.ncbi.nlm.nih.gov/pubmed/17477772
Review Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. [Lehmann U, Kreipe H., Methods. 2001] https://www.ncbi.nlm.nih.gov/pubmed/11846610
I am certain about other colleagues with valuable input will reply....best wishes and regards, WM.
If possible, I would use the range of samples fixed in 4% PFA for the different periods of time as controls. For example, the same liver samples fixated for 24h, 48h, 7days, 14 days and a month, in addition to your sample fixed for two months. Then for all the slides you should use regular antigen retrieval protocol (0.001M sodium citrate, pH 6 - 10 to 15mins on 95oC) and simply see what is the maximal period of fixation without masking antigens irreversibly. In consecutive slides, in parallel, you can try more aggressive antigen retrieval and compare to standard ones.
Good luck!
@Vukasin: IMHO very good (methodological) proposal....(:-)) Best regards, WM
I tink that the best thing to do is sercing the antigen on cryostatic section, to see its levels, and then tying with different methods and leghts of fixation. Use fresh tissue as marker.
If I did not misinterpret your question, if your goal is to detect antigen with immunocytochemical techniques and not DNA analysis, then over-fixation is not as big a problem as you might expect. If antibodies used for detection are properly titrated to give optimal detection, then for many, better formaldehyde fixation actually is better than less. For detection in paraffin of course you are combining fixative effects with extraction in alcohols and organic solvents which can sometimes affect antigenicity. A way of overcoming the fixative effects is to use epitope-retrieval (usually treatment with heat and either pH 6 or pH8 buffers) that unmask the epitopes needed for antibody binding. There are a number of papers describing how to do that and if coupled with careful adjustments of antibody concentrations when staining, are quite effective. Just heat and steam can work as well. When antigens are in low abundance, you should consider amplification of product (see Berghorn, K.A., J.H Bonnett, and G.E. Hoffman. cFos immunoreactivity is enhanced with biotin amplification. J. Histochem. Cytochem: 42:1635-1642, 1994. PMID 7983364)
see:
Antigen retrieval immunohistochemistry: review and future prospects in research and diagnosis over two decades.
Shi SR, Shi Y, Taylor CR
and
Perhaps another reading (interesting new aspect of a special fixation):
WEIGNER'S fixative.
For your convenience I've attached the pdf.
I am trying to figure out whether to process all of my animals which were sacrificed at different times all in one batch, or to process them immediately as they are sacrificed. I would like to compare cfos between them, but am not sure which method is more scientifically sound (i.e. whether or not cfos will degrade due to longer times in fixative for some if I do them all in one batch vs. comparing brain slices that were processed in different batches). Thank you.
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