Immunohistochemistry, the process of using antibodies to detect specific antigens within tissue samples, allows researchers to investigate the expression of protein targets on an anatomical basis. Providing insight to antigen abundance and localization, along with an indication of which upstream pathways may be activated, IHC relies on meticulous sample preparation to preserve target integrity and cellular morphology. Within a research setting, most IHC is performed with formalin-fixed, paraffin-embedded (FFPE) tissue. Produced by immersing excised tissue in a formaldehyde solution before embedding it in paraffin wax, FFPE sample preparation subsequently includes processes such as sectioning, antigen retrieval, permeabilization, and blocking, all of which require optimization to achieve meaningful, high-quality IHC data.
FFPE sections offer many advantages over frozen tissue
“The choice between frozen and FFPE sections really depends on the antigen you’re trying to detect,” explains Simon Renshaw, principal imaging scientist at Abcam. “Compared to FFPE sections, frozen sections display epitopes in a more native state. Some antibodies will only recognize the epitope in this state, making frozen sections essential in certain cases. However, if you can use FFPE sections they do have several advantages.” In addition to easy storage at room temperature, these benefits include elimination of antigen degradation through premature thawing (a process that can generate ice crystal artefacts upon subsequent re-freezing), better tolerance of heat-mediated antigen retrieval, and improved preservation of cellular morphology.
Optimal staining begins with fixation
“When you take tissue samples for FFPE IHC, it’s extremely important to fix them as soon as possible after excision,” says Renshaw. “Once normal homeostatic control is lost, antigen integrity and cellular morphology become compromised, the effect being greater over time and exacerbated by autolysis, hypoxia, and changes in temperature or pH. Tissue size and fixation time are key considerations at this stage, since the formaldehyde must penetrate the sample completely without causing over-fixation. Tissue should be only around 2–5 mm thick, with a surface area no greater than the cassette it’s being processed in, and it’s typical to perform fixation for around 12–24 hours. Over-fixation may introduce the need for extended antigen retrieval and can also cause the tissue to become brittle, making microtomy difficult.”
According to Jonathan Weinreich, associate product manager at Enzo Life Sciences, anything that may have a detrimental effect on microtomy is to be avoided. “Smooth and uniform tissue sectioning is essential to avoid generating misleading data,” he says. “If the thickness of the sections is non-uniform, it’s highly possible that a mis-match between experimental controls and test slides could occur.” Renshaw adds that if sections are too thick, light may struggle to penetrate the sample. However, Visikol, a company offering an innovative tissue clearing technology compatible with many thicker tissue specimens, says it has resolved this issue
Clearing facilitates imaging of thicker tissue samples
“The Visikol® HISTO™ technology was developed in collaboration with almost a thousand researchers worldwide to make tissue clearing a reliable tool for bio-imaging,” notes Michael Johnson, CEO. “Existing tissue clearing techniques require specific adaptations for differing tissue types, labels, fixation methods, imaging modalities, and custom microscope objectives, whereas our technology is easily applied to a wide range of tissue samples. Based on the process of refractive index matching, during which water is removed from the sample and replaced with a high-refractive index solution, clearing is conducted after fluorescent immunolabeling by incorporating just a few easy steps at the end of existing protocols.”
By rendering tissues transparent with Visikol HISTO, researchers can gain a far greater breadth of information without destroying cellular morphology. A further advantage of the technology is its reversibility—tissues can be imaged in 3D and then subsequently sectioned for follow-up histology. This allows easy validation for clinical applications.
Don’t overlook the intermediate processing steps
Equally as important as efficient fixation are the downstream sample processing steps, the first of which is to embed the tissue in paraffin wax. To achieve this, the sample must first be dehydrated through immersion in increasing concentrations of alcohol. “Paraffinization is a multi-step process that transitions the tissue from an aqueous (formaldehyde) phase to an organic (paraffin wax) phase,” notes Renshaw. “While it can be performed manually, experimental reproducibility can be greatly improved using an automated processing unit. These instruments help maintain antigen integrity and tissue architecture by using both heat and pressure to assist reagent impregnation.”
Once the tissue has been embedded and sectioned, antigen retrieval may be required. Antigens can often be masked as a result of fixation, making antibody binding impossible. “Although antigen retrieval is not always necessary, and depends on the antigen being demonstrated, this step is very important for accurate nuclear staining,” says Weinreich. “For most antibodies on the market, antigen retrieval is performed with an alkaline buffer solution (pH ~9.0), with many researchers choosing to avoid a proteolytic treatment since differences in fixation time can drastically affect staining. Antigen retrieval information is frequently stated on antibody datasheets, yet a literature search can often reveal useful information when this is not the case.”
Since tissue samples often contain endogenous components such as peroxidases, phosphatases, and biotin, which may interfere significantly with immunostaining, it’s essential that IHC sample preparation includes steps to block these biomolecules where necessary. When the immunostaining protocol is reliant on alkaline phosphatase- or horseradish peroxidase (HRP)-conjugated secondary antibodies for detection, endogenous peroxidases and phosphatases should be blocked with a reagent such as Enzo Life Sciences’ IHC Tissue Primer to avoid the generation of artefacts. When avidin-biotin or streptavidin-biotin detection systems are employed, avidin-biotin blocking systems should be used; these prevent false-positives due to the presence of endogenous biotin. Blocking of non-specific antibody binding sites should then be carried out ahead of any antibody incubations.
Upstream processing of tissue samples for IHC is equally as important as a well-optimized immunostaining protocol. Consider these tips to achieve effective sample preparation:
- Fix tissue samples as soon as possible after collection
- Ensure tissue sectioning is smooth and uniform
- Incorporate a tissue clearing step to render thicker samples transparent
- Consider an automated processing unit for paraffinization
- Research the need to perform antigen retrieval
- Block endogenous components to minimize background staining
Future advances
While FFPE IHC in its current format continues to see considerable utility, there is always room for improvement. The use of multiplex IHC with fluorescent dyes is an important growing area in immuno-oncology, according to Renshaw, enabling more thorough evaluation of spatial interactions of various antigens within tumor microenvironments. “The use of knock-out tissue for antibody validation is also becoming increasingly popular,” he says, “especially in light of concerns over antibody reproducibility. The integration of in situ hybridization alongside IHC to confirm gene expression in the same cells that exhibit antibody staining would also provide greater confidence in results.” Weinreich adds that the use of primary antibodies directly bound to polymeric reagents could also represent a significant advance in the field. “This would be a huge step forward, potentially allowing IHC protocols to be completed in under an hour.”