1. Why should I heat stabilize my tissue sample?
Heat stabilization increases the accuracy and preserves the quality of tissue samples by preventing enzymatic degradation of the biological components without the use of additives. This ensures a stable sample that will not change from the moment of heat stabilization until the point of analysis. This is especially beneficial for molecules that are highly sensitive to changing conditions (transient molecules) such as endogenous peptides, e.g. neuropeptides, and post-translational modifications, e.g. phosphorylated - and acetylated proteins, that typically degrade or change rapidly after excision.

2. How does heat stabilization work both in theory as well as in practice?
The heat stabilization technology utilizes rapid conductive heating to generate a fast, homogenous thermal denaturation of proteins. This results in complete, permanent denaturation, and thereby inactivation, of all enzymes (such as proteases, peptidases, phosphatases) that could cause further biological changes to the tissue sample ex vivo. Since the system is additive-free, i.e. do not require any addition of chemical inhibitors, potential interference with downstream analysis is removed. Biomarkers, peptides and posttranslational modifications such as phosphorylated and acetylated proteins are preserved close to their in vivo state throughout the entire workflow. Practically, a tissue sample is placed in the specifically designed Maintainor® Tissue card. The card is manually entered in the instrument, Stabilizor™ T1, and algorithms automatically determine the amount of energy required to ensure complete denaturation. Typically, a fresh tissue sample of the size of a mouse brain is heat-stabilized within one minute of heating. The heat-stabilized tissue sample may be further dissected, stored in the Maintainor Tissue card or analyzed.

3. Can multiple tissue samples be heat stabilized in the same Maintainor Tissue card?
Maintainor Tissue cards are designed to heat stabilize one tissue sample to ensure a consistent and reproducible preservation. Heating time is calculated based on the sample height and assumes physical contact with the sample with both upper and lower heating plates. If two tissue samples are placed in the same Maintainor Tissue cards, there is a major risk for the smaller sample to be subject to one-sided heating and thereby obtain only partial inactivation.

4. Is it possible to stabilize a tissue sample from a frozen state?
Yes, a frozen tissue sample is easily heat stabilized in the Stabilizor system. The frozen tissue sample is placed in a Maintainor Tissue card and the card is then entered in the Stabilizor T1 instrument and heat stabilized

5. How does the instrument ensure reproducibility?

  • Automatic algorithms calculate the energy required for each individual sample based on its height i.e. each individual sample is heated a specific time period. Consistent conductive heating of each sample is thereby ensured.
  • Enzyme activity is eliminated from the moment of heat stabilization.
  • Degradation is stopped permanently, without the use of additives.
  • The sample handling is standardized to improve reproducibility of analytical workflows
  • It enables linking upstream processing to downstream results, traceable treatment parameters are available
  • Facilitates accurate sample comparison and data interpretation downstream

Comparison of heat stabilization and other preservation technologies

6. Why is heat stabilization to prefer over using chemical inhibitor cocktails?

  • Heat stabilization is an additive-free inhibition and thereby is free from any toxicity and interference attached with them in downstream analysis technologies.
  • Heat stabilization assures complete inactivation of enzyme activity throughout the complete workflow whereas inhibition cocktails reduce the activity and is selective.
  • Heat stabilization is based on rapid, homogenous and permanent thermal inactivation of enzyme activity which assures high quality and reproducible data, no time is required for inhibitors to reach their specific targets.

7. Do you recommend using inhibitor cocktails during protein extraction on a heat stabilized tissue sample?
Inhibitor cocktails are not necessary to use during protein extraction on a heat stabilized tissue sample. Heat stabilization is an additive-free inhibition that assures a permanent and complete inactivation of enzyme activity throughout the complete workflow. Moreover, protein extraction of a heat stabilized sample can be performed in room temperature.

8. What are the differences in results using heat-stabilization compared to snap freezing?
Biological changes begin from the moment a tissue sample is removed from its native environment and, although conventional snap freezing may temporarily stop biological change, enzyme activity will return as a sample thaws during preparation for analysis. 
The additive-free heat stabilization technology increases the accuracy and quality of biological tissue samples from moment of excision throughout the entire workflow.

9. Is it possible to obtain the same results as with heat stabilization, if tissue sample is boiled or microwaved?
Boiling as a technology for sample preservation is a relatively slow process (because of insulating containers is needed to avoid contamination and diffusion) compared to heat stabilization in Stabilizor T1, thereby time is left for degradation to occur and that cause biological changes to the tissue sample. The head focused microwave as a technology for sample preservation generates an instant inhibition process and tissue stabilization of in vivo level. However the microwave technology is limited to fresh rodent brain tissue and has relatively poor reproducibility due to residual activity in local cold spots. Heat stabilization offers a standardized sample handling that improves reproducibility of the analytical workflow as well as electronic record/traceability of samples and is compatible with both fresh and frozen water containing tissue.

10. What are the differences in results using stabilization compared to freeze dying and /or lyophilization?
In heat stabilized samples enzymes are permanently inactivated whereas enzymes in freeze dried samples are only temporarily inactivated and may reactivate after rehydration and continue sample degradation.

Storage and stability

11. How should tissue samples be stored after heat stabilization?
Heat stabilized tissue samples are preferably stored in the Maintainor Tissue-cards in a -80°C freezer. This since there is other non-enzymatic processes such as oxidations that can affect the sample.

12. Will the morphology of the sample be affected after heat stabilization?
To achieve an efficient, rapid and reproducible heating the tissue will be slightly compressed. However, for most tissues the morphology is well preserved after heat stabilizing of a fresh tissue. The colours of the sample change and the tissue becomes more frail but fully anatomically recognizable. The morphology is somewhat more perturbed if a tissue sample is heat stabilized from frozen state.

13. What is the limitation of sample size possible to heat stabilize?
The heat stabilization technology is compatible with sample sizes up to D 30 mm and/or H 7 mm. There is no lower limit for tissue sample size.

14. How long time does it take to heat stabilize a tissue sample?
Typically a fresh tissue sample of the size of a mouse brain is heat stabilized within 1 min. A frozen tissue sample requires approximately 50% longer stabilization time.

Compatibility with sample type

15. What types of tissue samples are possible to heat stabilize?
The Stabilizor technology has been optimized for tissue samples such as; brain, muscle, liver, pancreas, xenograft, spinal cord etc. Other types of tissue such as bone may require optimization. If you have experienced potential post sampling change in other sample types e.g. cells, blood CSF etc. contact us for discussion and/or potential collaboration.

16. Some proteins, enzymes and phosphorylations are sensitive to heat, how does that comply with heat stabilization?
Heat stabilization is sufficiently rapid to inactivate heat-induced physiological responses. Thus, heat stabilization does not induce changes but preserve the sample.

17. Can freeze dried and/or lyophilized samples be stabilized?
No, efficient heat stabilization require hydrated samples.

Compatibility with extraction and downstream analysis

18. Which analysis techniques are compatible with heat stabilized tissues?
After heat stabilization, of either fresh or frozen tissue samples, in a Stabilizor system, biological components can be extracted and analyzed using common buffers and techniques. Compatibility with a number of downstream analytical techniques has been verified, including mass spectrometry, phospho-shot gun, MALDI Imaging, Western Blot with phospho-specific antibodies, 1D and 2D gels with phospho-specific stains and reversed phased protein arrays (RPPA). See also FAQ (number 24) for recommendations how to achieve most efficient extraction of proteins from heat stabilized tissue.

19. Are there any considerations when extracting analytes from heat stabilized samples?
To achieve the most efficient extraction of proteins from heat-stabilized tissue it is of high importance to follow Denator´s developed technical note “Extraction of proteins from heat-stabilized tissue samples”, see highlights below;

  • Use a denaturing buffer (>8M Urea, >1% SDS, >6M GuHCl, SDS)
  • Use a buffer-to-sample ratio greater than 10 (>10μl buffer/mg sample)
  • Ensure thorough homogenization using ultrasonication, grinding or ball mill

The extraction procedure can be performed in room temperature which streamlines the tissue workflow.

20. Does the Stabilizor system also inactivate kinase and phosphatase activity?
Yes, all enzyme activity is stopped including that of kinases and phosphatases. This enables quantitative proteomics, peptidomics and phosphorylation studies.

21. Is it possible to analyze other post-translational modifications (PTMs) than phosphorylations? Can heat stabilization be used as  a general stabilization method of PTMs?
Our customers have demonstrated that phosphorylation states, sumoylation states, as well as acetylation states are preserved after heat stabilization.

22. Is it possible to measure enzyme activity on a heat stabilized tissue sample?
No, it is not possible to measure enzyme activity on a heat-stabilized sample since all enzymes are inactivated due to denaturation.

23. Is it possible to analyze natural and drug metabolites from a heat stabilized tissue sample?
Yes, it is possible to analyse both natural and drug metabolites from a heat stabilized tissue sample. Both classes of metabolites are potentially affected by post-sampling processes and can benefit from heat stabilization. Metabolites are a diverse class of molecules and relatively little research has been done to study the effect of heat stabilization so special considerations and optimization may be necessary to ensure optimal benefits.

24. Is it possible to extract and analyze DNA and RNA from a heat stabilized tissue sample?
Yes, It is possible to extract and analyze DNA and RNA from stabilized samples. mRNA is intact and can be used as would you in any other type of sample. However, due to residual RNase activity even in stabilized samples, RNA will degrade unless additional measures are taken.

25. Are there any considerations when preparing sections MALDI imaging from heat stabilized samples?
Yes, heat stabilized tissue samples will appear more brittle and cryosectioning will require special attention. Read more on how to ensure good morphological preservation in sections from heat stabilized tissue, or contact a Denator application specialist for further information inform@denator.com.

Highlights from MALDI Imaging application note;

  • Full length endogenous Pep-19 at higher levels in stabilized samples.
  • Ex vivo produced Pep-19 fragments only detected in snap frozen samples not in stabilized samples.
  • Protocol for good section quality from stabilized tissue.

26. Will the tissue sample size, shape and general appearance change after heat stabilization and will it have any implications on MALDI Imaging analysis?
Since the heat stabilization technology is based on conductive heat the sample and the overall tissue dimension may be somewhat compressed. The colors of the sample change and the tissue becomes more frail but fully anatomically recognizable. Read more in the guideline on how to ensure good morphological preservation in sections from heat stabilized tissue for MALDI Imaging analysis, or contact a Denator application specialist for further information inform@denator.com.