Tissue processing - how to succeed with your IHC part I

Would you print your childhood best and precious memories on a low-quality photographic film? I wouldn’t. However, just like me, you probably have hundreds of old faded yellow photos abandoned in a drawer. Images quality relies on high-quality printing films…and so does your immunohistochemical staining. Tissue handling and tissue processing determine the quality of your IHC results. You want high resolution?  Let’s understand how to achieve it.

How incredibly tedious and frustrating it is to spend countless hours on an IHC only to find that you produce a low-quality staining, almost microscopically uninterpretable staining? Would you start blaming the quality of the antibody, the IHC protocol, or make a call to a service technician responsible for maintaining and repairing the microscope?  Wait, those factors are not always the source of the problem. Have you considered the quality of your sample? The way the tissue has been prepared strongly impacts the results. Read on to opt for a technician-free solution. At the end of this post, you can download our checklist to keep track of your IHC experimental conditions.


The Importance of Tissue Handling and Tissue Processing

The quality of the staining, resulting from the antibody-antigen binding, is to a variable degree affected by alterations during tissue handling and tissue processing. Some proteins are most susceptible to changes in fixation than others. The most common cause of poor immunostaining is not the technique itself but the potential influence on the antigenic sites during tissue preparation. 


The Journey of a Specimen: From Patient to Section

There are many reasons to examine cells and tissues under the microscope: for basic and clinical research, to evaluate potential biomarkers in clinical patient cohorts, from small projects to high-throughput strategies.

Human or animal tissue - whether biopsies, larger specimens removed at surgery or tissues from autopsies - taken for diagnosis of disease or for research purposes must be processed in the histology laboratory to produce microscopic slides that can be interpreted microscopically. They range from very large specimens or whole organs to diminutive fragments of tissue. 

The specimens to be processed are usually solid, heterogeneous and irreplaceable. For this reason, it is imperative to avoid mistakes since a damaged or unusable specimen can lead to an ambiguous result or misdiagnosis.


Getting the Best From Your Tissue

The quality of a sample is generally based on the microscopic assessment of the histology of the tissue often using routine H&E-staining.  Due to the diversity in specimens and large range of clinical inquiries, the procedures within the histological laboratory are very diverse. There are however common tissue processing steps among the histological procedures that impact the quality of the results independently from the specimen type.


Factors Affecting Tissue Integrity

In addition to the time, temperature, and type of fixation, multiple other factors during tissue collection, handling and tissue processing may affect histological or immunohistochemical staining. 

  • Mechanical artifacts caused by improper tissue handling include shrinking, swelling, grinding (due to an abrasive cutting), crush artifacts, diffusion of unfixed material (streaming), solubilization of hydrophobic cell components, loss of protein tertiary or secondary structure, antigen masking. Those fine cracked lines that you occasionally may observe within the tissue are due to a defective blade during tissue sectioning. So, always make sure to use high quality, sharp blades for cutting. Knife tilt angle must be optimized for each microtome and blade type. 
  • Biological artifacts include tissues features and modification such as necrosis, hemorrhage, and inflammation, and biological processes within the tissues might all affect the staining performance. Diseases have a great impact on tissue quality, not least within liver, pancreas and skin.
  • Experimental artifacts such as storage time, incubation time, buffers and consumables (plastic or glass) may have an influence on the tissue sections and affect the results of IHC. Be aware of the shelf life of the reagents you are using; they cannot be used for an indefinite period. Some require refrigeration because they are inclined to support microbial growth. Replace regularly solvents and staining reagents based on the number of slides stained or racks processed. For tissueprocessing, the most important problem is in fact, the inadequate tissue dehydration prior to paraffin embedding. This can be prevented by preparing all solutions freshly every week, depending on the volume of tissue If consistently applied, these procedures could eliminate some of the sources of variation in immunohistochemical stains.



Uncontrollable and (Potentially) Controllable Variables

The first steps in sample collection, handling and tissue processing, impact the quality of the results. These steps often challenging to control, are classified into those over which you, the investigator, have no control, such as the patient’s history or the events occurred during the biopsy prior to receipt of the tissue (uncontrollable variables, Figure 1) or those that you can potentially control after receiving the tissue, such as the experimental protocol, including buffers and consumables (potentially controllable variables, Figure 1).



Figure 1. Schematic of potential sources of uncontrollable and controllable variables during tissue collection, handling and tissue processing.


Do You Know What Staining to Expect?

 Since the expression levels of protein differ widely in different tissues, there is no protein that can be used as a universal marker for the overall quality of an IHC staining. 

 For this reason, you must know what you are trying to demonstrate with the staining you are performing. Just “following the protocol” and not really knowing what should be seen in the finished section will lead to poor results.

Always ask yourself: “Where I am expecting to see the staining? Is the protein expressed in my tissue? Does my antibody work in fixed tissue? Which molecules/structures did I preserve: proteins, nucleic acids, polysaccharides, lipids or other features?”


“Routinely Fixed and Processed”?

Avoid writing "routinely fixed and processed" in your next manuscript. There is no standard tissue processing protocol for the optimal demonstration of all antigens. Histological and histopathological procedures depend entirely on the type of experiment and has to be worked out empirically. 

Factors involved in all aspects of tissue preparation can affect tissue quality and hence the histological results. It is important that all details of the preparation schedule and tissue processing are given when reporting immunocytochemical studies, rather than using the general terms.


The Biology Must be on Display, not the Technical Artifacts!

Even though some artifacts can be avoided, some even reverted, others can be compensated for, always make an effort and try to avoid them. 

Treat your tissue like the canvas for your best image so that when you will open the drawer in to look at your staining in some years you will see bright and clear colors and not a yellow faded image.


Download and print our free useful checklist to keep track of your IHC experimental conditions.



Recommended Reading

Hewitt S.M. et al. (2008), Tissue Handling and Specimen Preparation in Surgical Pathology: Issues Concerning the Recovery of Nucleic Acids from Formalin-Fixed, Paraffin-Embedded Tissue, Archives of Pathol & Lab Medicine, Vol. 132 (12), pp.1929-1935.

Lawrence D. True (2008), Quality control in molecular immunohistochemistry. Histochem Cell Biol. Sep; 130(3): 473–480.




Written by Dr. Kristian Moller

Dr. Kristian Moller is a Principal Scientist at Atlas Antibodies. He holds a Ph.D. in Molecular Neurobiology from the Medical Faculty at Lund University Sweden. Kristian has a profound national and international R&D experience as a specialist in applied molecular histology from the private pharmaceutical, diagnostic and immunotherapy sectors. There his work has emphasized on research devoted to tumor diagnostic antibodies, T-cell mediated cancer immunotherapies and early drug discovery within dermatology. In addition to his general expertise in tissue biomarkers, he is strongly specialized in the technical aspects of immunohistochemistry and RNA in situ hybridization.

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