AMP Core Lab – Technical Information
Artifact or appearance of formalin-fixed paraffin embedded tissues
- Crushing of cells or tissue – mechanical distortion during handling prior to fixation
- Understaining and/or poor morphological detail – usually inadequate or delayed fixation
- Overstaining – usually suboptimal fixation, but sometimes thick sections or staining problems
- Overstaining plus distortion of nuclei in surgical specimens – surgical electrocoagulation artifacts
- Fixation pigments on tissue – suboptimal processing
- Fragmentation of sections – usually suboptimal fixation or tissue calcifications, but sometimes a nicked microtome blade
- Extraneous tissue present in multiple slides cut from a single paraffin block – contaminant introduced during handling prior to embedding.
- Extraneous tissue fragments present in only a single slide – usually represent “floaters” from a contaminated water bath
Fixation of tissues for histopathological analysis
The preparation of tissues for histological examination is more complex than often appreciated. The quality of sections and stains can be influenced by many factors. Although some factors involve the processes of paraffin embedding, sectioning, or staining in the histology lab; many others are related to prior tissue handling.
In most contemporary histology labs, fixation is the single most important determinant of high quality histological sections. Consistency of most post-fixation steps is facilitated by appropriately maintained automatic tissue processors, embedding centers, and autostainers. As a result, problems related to the quality of stained sections are more often related to inadequate fixation than to problems with processing and embedding. Most sectioning artifacts can also be attributed to problems with fixation. The following information is provided for investigators, clinicians, or trainees who may not have prior training in histology or anatomic pathology.
Fixation with formalin
The most widely used fixative for routine histology is 10% neutral buffered formalin (NBF, approximately 4% formaldehyde). This fixative can effectively prevent autolysis and provides excellent preservation of tissue and cellular morphology. Other fixatives may be preferred for certain types of assays. However, 10% NBF is also considered the fixative of choice for many other procedures that begin with paraffin embedding, including immunohistochemistry, interphase FISH, and next generation sequencing.
Important Variables – As discussed below, many variables must be carefully controlled to insure consistency and reproducibility of fixation. These include the:
- time interval between tissue harvesting and fixation,
- composition of the fixative,
- volume ratio of tissue to fixative,
- duration and temperature of fixation,
- tissue thickness,
- conditions of any post-fixation storage, and
- the interval between fixation and embedding.
Every attempt should be made to define each of these variables for any experiment, and particularly for any series of experiments. Although the process is reasonably straightforward, a detailed written protocol is recommended, and personnel need to receive sufficient training to understand and reproduce the procedure, thereby avoiding inconsistent results. Investigators or trainees are encouraged to interact with lab management prior to initiating any studies to discuss their objectives and confirm a suitable protocol. For basic research studies, it may be desirable to perform preliminary experiments (e.g. various fixation times), prior to committing tissues from large numbers of valuable experimental animals. For clinical research, conditions for routine clinical testing should usually be used.
Influence of Tissue Type – There is no single, optimal procedure that can be used for all types of tissue. As indicated below, the presence of abundant blood or fat, fibrous capsules, and skin can greatly slow tissue fixation. In addition, longer times are usually needed to fix loose non-fibrous (e.g. fetal or embryonic) tissues.
Bony or densely calcified tissues can be particularly problematic. Such tissues need to be completely fixed before decalcification. Although decalcification almost always results in some loss of cytological detail and/or alters staining properties, decalcification of incompletely fixed tissues almost invariably leads to unacceptable results.
Variables that influence formalin fixation
Post-Mortem or Post-Surgical Interval. Autolysis begins immediately following disruption of blood flow and rapid fixation is essential. Because autolysis can influence the cellular morphology and staining, the time interval between death and fixation should be minimized and controlled whenever possible. If there is a necessary delay in fixation, the tissue should be immersed in cold phosphate-buffered saline (PBS). Tissues should not be allowed to dry before (or after) fixation. For certain studies, vascular perfusion with fixative may be recommended.
Fixative Composition. Formaldehyde fixation is usually optimal near physiological pH and ionic strength. Unfortunately, formalin is not stable and will gradually acidify and form more complex polymers, with adverse effects on fixation. This is minimized by appropriate buffering and inclusion of small amounts of methanol. Nevertheless, formalin has a finite shelf-life. Commercial preparations in specimen containers are strongly recommended, and should not be used beyond the expiration date, usually 2 years.
Fixative Volume. Tissues should be immersed in a >20-fold volume excess of fixative. Large amounts of blood or protein-rich fluid can decrease the effective fixative concentration. If necessary, this problem can be minimized by brief washing of the tissue with PBS prior to placement in the fixative. If the fixative becomes obviously cloudy or bloody the fixative should be changed.
Fixation Time. Fixation with 10% NBF at room temperature usually provides excellent morphological detail. For most animal tissues, a minimum of 24 hours at room temperature is recommended. However, some bloody or fatty tissues, and certain fetal tissues, could require significantly longer fixation, e.g., up to 48- 72 hours. If sectioning of a fixed tissue reveals internal areas with normal coloration, the fixation time should be extended. Small clinical biopsies are sometimes fixed for only 6 hours, but 12 or more hours will often provide a better result.
As suggested above, under-fixation is more of a problem than over-fixation for most histochemical procedures. Under-fixation can be associated with artifacts secondary to alcohol exposure during tissue processing, or tissue damage during decalcification procedures. Over-fixation can impair staining, and complicate sectioning, but can more often be compensated for by the histotechnologist.
Fixation Temperature. Fixation time and temperature are related variables. An increase in temperature can increase the rate of fixation but can also increase the rate of autolysis. Primary fixation in the cold can slow autolysis, but also slows the process of fixation, and premature cooling of a specimen in fixative can lead to inadequate fixation.
Tissue Thickness. Formalin fixation is dependent on diffusion of the fixative into the tissue. The thicker the tissue, the more time is required to obtain complete penetration and cross-linking of tissue proteins. If the specimen is too thick, the center of the tissue can suffer from autolysis or incomplete fixation. For this reason, tissues should be no thicker than 3 mm.
Careful slicing of tissues and solid organs prior to transfer to fixative can greatly influence the efficiency of fixation by increasing exposed surface area and decreasing total thickness. This is particularly important for fatty, bloody, or organs surfaced by a capsule or membrane. When slicing tissues, it is important to use a sharp, clean blade to minimize compression artifacts. If forceps are used to stabilize the tissue, care must be taken to avoid applying excessive pressure. For very soft tissues, it is sometimes helpful to stiffen the tissue by brief fixation prior to cutting of thinner sections and additional fixation. Tubular organs can be cross-sectioned at small intervals to enhance exposure of the lumen to the fixative; any luminal contents should also be removed.
Post-Fixation Storage. Non-coagulant fixatives such as formalin continue to cross-link proteins as long as they are in contact with the tissue. If there is to be a delay in processing after complete fixation (usually 24 hours or more), tissue can be stored for up to 3 days in the cold in 70% ethanol. However, it is essential that the tissue is completely fixed prior to transfer to the alcohol. Premature transfer of incompletely fixed tissue to alcohol precludes normal tissue processing has been reported to permit time-dependent reversal of cross-linking.
Tissue fixation is one of the most important determinants of the quality of histological sections. Incomplete fixation can lead to unsatisfactory and poorly reproducible results. The effects of inadequate fixation can sometimes be partially compensated for, particularly if recognized before embedding, but they cannot be fully reversed. It follows that the best results often require a close interaction between the investigator and Histology Lab. If you have unsatisfactory results, please bring the specimens to the lab for review with the Lab Supervisor or Manager. If you have any questions, do not hesitate to contact the lab staff
Immunohistochemical procedures and reagents
Ventana Medical Systems
Ventana has a single URL and complex navigation. To find an antibody on the site click on Products > Online Catalog > Primary Antibodies. To download a datasheet you need to enter the catalog number in the search box for the Product Document Library.
Cell-Marque is the source of many of the Ventana antibodies. However, the catalog is easier to navigate and often more complete. Antibodies available in Ventana dispensers are specifically indicated.
Simplified description of TMAs
Antibody or probe optimization
Antibody selection guide
The following document was prepared for the AMP Core Labs by Dr. Erika Crouch. Please feel free to forward comments or suggestions.
Most selection criteria are generally relevant to immunohistochemistry (IHC) using formalin-fixed and paraffin embedded tissues (FFPE). However, some are more specifically relevant to the Ventana autostainer used by the Core Labs. Although this document emphasizes the general properties of antibodies (Ab); issues of antibody specificity are the focus of the accompanying document and checklist.
Many commercial antibodies and in vitro diagnostic tests only demonstrate their advertised properties in the context of a specific tissue or assay platform. Some well-recognized complexities of IHC using FFPE include:
- Abs can show specific interactions with the mature protein in blots or other assays, but still lack sensitivity or specificity when applied to complex tissues;
- Abs that show a single reactive species in blots of one complex tissue can show unrelated cross-reacting species in another.
- Specificity can differ between fixed and unfixed tissues because of unpredictable effects of fixation and embedding on specific and nonspecific Ab binding.
- Even if an Ab shows the expected pattern of reaction in assays using the recommended positive control, different patterns of reactivity can be observed in the context of other tissues or disease states.
Criteria for antibody selection
The first step in Ab selection is to carefully compare the manufacturer’s data sheets and associated primary literature for the competing products. There is no gold standard approach. However, the collected information can help identify the most suitable reagents. Although a systematic comparison of reagents takes time, it is almost always time very well spent.
- Preference should be given to Abs known to specifically react with the protein in FFPE tissue. If the manufacturer does not indicate suitability for IHC, there is probably a reason.
- Mouse or rabbit monoclonal Abs (MoAb) or rabbit polyclonals are preferred for the Ventana platform. Other primary Abs may require manual assays or additional optimization.
- MoAbs are often preferred over polyclonals, but can have their own limitations. MoAbs recognize a specific epitope, but this does not mean that they are protein specific. In addition, some MoAbs possess charge or hydrophobic properties that result in remarkably specific appearing non-specific reactions.
- Because MoAbs recognize a single epitope, they are sometimes less sensitive than a polyclonal Ab, and may not be usable for fixation-sensitive epitopes.
- Preference should be given to highly purified Igs or affinity-purified Abs, which can then be mixed with purified blocking proteins or other defined reagents. Whole serum, crude serum fractions, ascites, or unfractionated culture supernatants often show nonspecific reactions, particularly with low titer antibodies. For example, natural or autoantibodies in antisera can lead to nonspecific staining.
- Antigen purity is often critical for polyclonal Abs. Although specificity can be enhanced by affinity purification, effectiveness depends on the purity of the antigen coupled to the affinity column. Many affinity-purified polyclonal Abs are not specific for the putative antigen.
- Most commercial MoAbs are IgG1 and IgG2 and the core has appropriate control Igs. Studies with other isotypes will often require the investigator to obtain the corresponding isotype control.
- Preference should be given to high titer Abs. Low titer Abs are more often associated with background problems. They also increase reagent costs and can preclude migration of the assay to a high-throughput automated platform. Ventana dispensers are designed to accommodate relatively large volumes and there is a significant hold back volume (100 microliters) that limits complete utilization of the reagent.
- If a synthetic polypeptide was used as antigen, it is important to critically examine the sequence to avoid confounding specific cross-reactions. Have homology searches been performed against available human databases? Is the parent protein a member of a known protein family and/or is the sequence homologous to sequences found in unrelated proteins? Preliminary homology searches (e.g., with BLASTP) are recommended for all Abs using peptide antigens.
- When possible, studies of human tissues should employ Abs to human antigens or sequences. Specificity in one species does not insure specificity in another. Even highly homologous antigens can show significant differences in interspecies cross-reactivity.
- When possible, preference should be given to MoAbs with known epitopes. It can be helpful to know whether the epitope is sequential or conformational; whether reactivity is dependent on disulfide bonds, divalent cations or other factors; and the predicted location of the epitope on the native protein. Sequential epitopes may be more readily detected in FFPE. On the other hand, a buried sequential epitope that requires denaturation or sulfhydryl reduction for detection by Western blot may be masked in the tissue.
Criteria for antibody specificity
The following checklist was prepared for the AMP Core Labs by Dr. Erika Crouch to facilitate comparisons among antibodies. Please feel free to forward comments or suggestions.
There is no single approach for establishing antibody specificity in immunohistochemical assays. However, combinations of approaches can often provide convincing evidence. The more checkmarks the better! How does your preferred antibody stack up?
Lab safety checklist form