FAQs

There are pros and cons to both chromogenic and fluorescent immunostaining methods. Chromogenic IHC can provide a scientist or pathologist more morphological context that can enhance interpretation. Moreover, chromogenic immunostaining is not light sensitive, and labeling will tend to not degrade over time or with exposure to light. Fluorescent immunostaining can be better in instances when it is necessary to co-stain for two or more targets, and a high level of co-localization is expected. The appropriate staining method will depend on the goals of the specific project.

Many factors can affect ISH staining. Delayed fixation can cause RNA to degrade, and under-fixation can cause over-digestion of the tissue and result in poor morphology. Conversely, over-fixation can result in under-digestion of the tissue and result in no staining. Sample age can also affect RNA degradation, and slides should be cut fresh (<1 month) before ISH staining.

Generally, FFPE tissue is preferred, as the tissue morphology is better preserved and the sample is more shelf stable and not as susceptible to fluctuations in temperature. However, frozen tissue can be better in some circumstances. For example, formalin fixation can sometimes mask antigen epitopes; therefore, some antibodies may not be compatible with paraffin processing even with antigen retrieval. In these cases, frozen tissue may be a suitable alternative.

ISH and IHC staining methods can generally be combined easily as long as no protease steps are required in the ISH protocol.

Tissue should be fixed in formalin for no longer than 48 hours to prevent over-fixation. Most tissues can then be transferred to 70% EtOH or PBS + 0.01% Azide for long term storage. However, CNS (central nervous system) tissue should never be transferred to EtOH as this tissue is more sensitive to dehydration and should be transferred to PBS + 0.01% Azide.

Optimal IHC conditions will vary with each antibody, and an assay development phase is required for all new targets. A good starting concentration is 10ug/ml, which can then be adjusted up or down based on the labeling intensity and degree of non-specific background staining. Testing multiple antigen retrieval conditions, including enzymatic and heat-based retrievals, is recommended in all assay development phases.

Slides stained with chromogenic IHC or ISH methods can be stored at room temperature in a dry place, preferably in a slide box. Slides stained with fluorescent IHC or ISH methods should be stored in a refrigerator (4°C) in an opaque slide box to prevent exposure to light.

Choosing fluorophores will depend on the filter sets being used to visualize staining. Some filter sets overlap spectrally with others and require spectral unmixing software to distinguish. The following filter sets are the ones that we have found to be the most compatible with the least amount of spectral overlap: Dapi, FITC, TRITC (Cy3), Cy5, and Cy7. The FITC channel will generally demonstrate the most autofluorescence in FFPE tissue and should be reserved for the highest expressing target in the panel.

Preventing autofluorescence in FFPE tissue can be very difficult. Many commercially available autofluorescence quenchers are available, but the performance of such quenchers can vary. Generally, the best way to prevent autofluorescence is to use high quality antibodies with relatively intense expression so that exposure time when imaging can be minimized. Additionally, perfusing animals prior to tissue collection can reduce the number of erythrocytes, which are notorious for producing autofluorescence in most channels.

Yes! While you may come to us with specific interests in mind, we may offer suggestions for staining paradigms that have worked well in the past or will be more optimal for your endpoints of interest. We'll also advise on which pathology or image analysis methods will provide you with the most meaningful datasets. We offer staining/analytical pilots to help foster understanding of our process and can work with you to identify what will be most informative for your study goals.

We serve small-large pharma/biotech, start-ups, and academia.

Setting up a CDA prior to the consultation allows both parties to share critical information to guide the discussion. However, we can absolutely chat without an agreement in place and move forward when you’re ready.

Yes! We can accept materials at any stage after fixation and can provide any deliverable from our lab’s service areas: blocks, histochemically or immunohistochemically-stained slides, whole slide images, manual or digital pathology data and reports.

We have sites in both Boston, MA and Dallas, TX.

Big Picture: What is the goal of your research? What diseases/therapeutic areas are you focusing on? What is your drug platform? What level of involvement do you need from us: reliable service provider to invested collaborator? Study Timeline: What stage is the study at, i.e. are you planning the in-life? Has it already begun? Are materials collected? Deadlines: Do you have a specific deadline to meet for a high-level meeting/grant submission? Strategy: Is a stepwise approach helpful? Do you have references for endpoints? Are there specific endpoints that are critical for your regulatory submission? Do you have specific features that need prioritization?

We love to get to know you and your projects well before histopathology is being considered. This allows us to offer advice on model selection and lend insight into endpoint considerations. We can also connect you with any one of our partnering in-life CROs and consultants so you can start off on the right foot.

We cut routine sections at 4um. However, for some special stains we will cut them at 8um (ex/Toluidine Blue)

Under fixing tissue can result in autolysis and protein degradation. It may also cause staining artifacts. The size of your tissue samples and the amount of fixative used will greatly affect the amount of time needed for proper fixation.

• 10% neutral buffered formalin (preferred), use 1 part tissue to 15-20x amount of NBF or 4% paraformaldehyde (similar to 10% NBF)• 70% ethanol for cytology specimens (not recommended for central nervous system histology)

In general, we try to avoid keeping samples in formalin past 48 hours to prevent over-fixation. Over-fixation can mask epitopes and reduce antigenicity. If you are planning on doing IHC/ISH, then samples should be transferred into 70% ethanol or PBS at 4º C after fixation. However, we have successfully run studies on tissues that were stored long term in formalin. One exception is brain/CNS which should never be placed in ethanol as this tissue is sensitive and easily susceptible to drying out.

• Properly labeled containers with animal ID and study number. Animal ID needs to be properly labeled twice on the container if possible, in case one gets wiped off.• Be careful of collection artifact (ex/ crush artifact from forceps)• Gently stretch tissue as you lay it out (ex/ mouse GI, rat skin) to avoid scrunching up of tissue. Consider instead: Skin samples may be allowed to lightly adhere to a piece of cardstock with the subcutaneous side down before fixation to ensure good orientation for histology.• Place tissues in fixative as soon as possible to reduce autolytic effects.• Avoid letting the tissues dry out during necropsy.• We typically use Revised guides for organ sampling in rats and mice - Part 1 (nih.gov) or the website version Revised guides for organ sampling and trimming in rats and mice (fraunhofer.de) as a reference

To avoid the specimen defrosting during shipment please place all specimens on dry ice (blocks or pellets) and use an insulated box for shipping.

Yes. Morphologically, the tissue will be fine and there will be no issues if you are requesting H&E or special stains. If you are requesting IHC/ISH then over-fixation can mask epitopes and reduce antigenicity. It is best to consult with the advanced staining team to get the best results out of your tissue.

• Fix for a MINIMUM of 24 hours; aim for 48 hours as a standard• IHC/ISH – no more than 48 hours – must transfer to alcohol (70% ethanol)• Livers/spleen/dense tissue: 36-48 hours• Cells/cell pellets: 15 min-48 hoursNote: Samples need to be fixed in containers 15-20x volume of the specimen and NOT overcrowded in the container. If the specimen is biohazardous, contact us for fixation recommendations

Samples for cryo work can be sent as unfixed or fixed tissue frozen (by itself) or frozen (embedded in OCT). If you are submitting frozen samples already embedded, please do not use excess OCT as this may affect mounting of the samples. We cannot perform cryo work on paraffin processed samples.

Specimen jars/containers need to be secured to avoid spillage of fixative and specimen not drying out during shipping. You can wrap parafilm along the lid and/or place containers in a plastic bag. Put the entire thing in a padded box for protection. Please remember to send a sample list of your specimens in the box or via email.

After appropriate fixation, place specimens in Immunocal (a mild formic acid decalcifier) for 1-3 days depending on size. For samples destined for RNA work, we recommend instead an EDTA based decalcification solution (2-4 weeks on a shaker). In the end, your tissue should be rubbery and easy to bend.

Place slides in a slide box with folded paper towels or other thin packing material on top of the slides to prevent movement in the slide box during shipping. Tape around the slide box to ensure the box doesn’t open during shipping. Then place the slide box in a padded packaging box along with sample list of slides.

Yes, we accept blocks prepared at other locations. Once received, we will examine the tissue in the block and determine if the orientation of embedding is correct. If necessary, we will re-embed the samples.

No, Oil Red O can only be performed on cryosections. Oil Red O is a special stain that detects lipid content in tissues. The processing procedure used to create paraffin blocks will remove fats from the adipocytes. Oil Red O staining can be performed on formalin-fixed tissues as long as they have not undergone tissue processing or contacted ethanol or xylene solutions.

PSR/MT are acceptable for assessing collagen in heart, lung, liver, and kidney, though PSR is preferred. We recommend collagen IHC for GI and for any subtle changes expected.

After you will decide what features you want to see on the slide and appropriate trimming is done, generally, cut side down is how specimen tissue is embedded. We can assist you with your trimming/embedding to ensure we meet your research goals/needs.

We can accept samples in formalin, however, depending on the shipping carrier regulations you may need to transfer your samples into a different solution for transport. All types of tissues can be transferred to 70% ETOH or PBS for shipping EXCEPT for CNS samples.

The study protocol is the single best piece of information to share with the pathologist. If not available, a sample list and the experimental design, including model induction procedures, the number of groups, treatment information, and dosing paradigm are adequate.

Our pathologists and digital pathology team can discuss options for various endpoints to best suit your needs; however, some features are routinely analyzed by image analysis, such as quantifying cell/structure numbers, area of tissue affected, or intensity of a histochemical or immunohistochemical stain. Regardless of the type of evaluation, a pathologist will review the slides, interpret the data, and provide contextual information in the report.

We use Visiopharm to perform all of our quantification.

At minimum, an efficacy study should include 1) a model control (no disease induction) of the same age, strain, and sex as animals used in the study, and 2) a vehicle control with disease induction. These two controls allow the pathologist to determine the extent of disease induction compared to any background (non-model-associated) findings.Safety studies should always include a vehicle control of matched age, strain, sex, and administration route to allow for determination of test article-related findings.Our pathologists are happy to review study protocols and can answer questions regarding experimental designs.

We regularly accept and evaluate human tissue samples of nonclinical origin in addition to tissue from laboratory animals. Our veterinary pathologists can help answer non-diagnostic questions (e.g. cell type identification or immuno histochemistry interpretation) in human tissue. An MD pathologist is available to assist with questions that require diagnostic interpretation.

We are flexible and can provide a deliverable best-suited to your needs and timeline. We provide brief pathology reviews and datasets only on a limited basis, but all analyses and their quantified data are overseen by a pathologist regardless of formal reporting or not.

Yes, we have a selection of proofs of concept materials we have generated to help you visualize the endpoint and how that is interpreted by a pathologist. Please inquire if you have any specific needs outside of our sample data – we offer pilots to help foster understanding of our process and can work with you to identify what will be most informative for your study goals.

For most studies, blinded evaluation is not requested or performed.Masking of group/sample identities can be useful for preventing bias in studies with a clear hypothesis. For example, “Treatment A reduces the severity of inflammation in the DSS model of colitis.” If blinding is desired in such a case, we typically mask group identities, which are revealed following the evaluation, for appropriate reporting. For non-hypothesis-driven studies, such as toxicologic pathology/safety studies or model development, bias is desired. The pathologist’s role is to identify all possible findings and masking of group identities significantly limits the pathologist’s ability to do so. As such, blinding in this context is strongly discouraged.

An ACVP board-certified veterinary pathologist will evaluate nonclinical studies. If desired, an MD pathologist is available to evaluate human tissues. For efficacy studies, the pathologist will help choose the best scoring scheme for each model. Microscopic findings will be scored according to severity, following best practice guidelines. A published scoring scheme may also be utilized or modified to fit specific features of interest. For toxicologic pathology/safety studies, findings will be diagnosed according to standardized nomenclature and scored according to best practice guidelines from the Society of Toxicologic Pathology.

Yes! We perform a quality control check of all submitted materials and will inform you if additional materials, such as the tissue blocks, are required to meet your evaluation goals.