Proteintech strives to validate all of its antibodies in multiple applications to make your job of using them that bit easier. It has a department dedicated to the assessment of its antibodies for use in Immunohistochemistry; the work of this team is devoted to ensuring Proteintech’s antibodies have IHC data available for potential and regular customers to view on its website (www.ptglab.com), and most importantly that these data meet current literature standards.
As someone whose research background in IHC doesn’t stretch much further than reading about it and viewing results in the academic literature, I thought it was high time I learned a little more about the practical side of the application; so, on my recent visit to Proteintech’s laboratory facilities I spent some time with the IHC team, looking to get to know the ins and outs of IHC validation a little better…
Soon after my arrival at the lab Joanna King, head of the IHC department and my guide, first tutors me on the selection of tissues for IHC. The team takes time and care over searching for either physiological or pathological representations of tissues suitable to test the antibodies due for IHC validation. Tissues are mainly selected for their expression of specific target proteins; for example, if an antibody has been raised using an antigen closely associated with a certain cancer, tissue samples from these types of tumor are sourced. For instance, the overexpression of mucin 1 (MUC1/CA15-3) is documented in epithelial tumors such as breast and ovarian cancer by the literature – so Proteintech’s polyclonal MUC1 antibody (19776-1-AP) has been tested in IHC in these tissue types, and the results are displayed on its website.
All tissues used in the IHC lab are formalin fixed paraffin embedded (FFPE) sections; these are the standard used by in-vitro diagnostic (IVD) and pathology labs, where samples must be stored long-term. I instantly understand the significance of this choice as the nature of formalin fixation and paraffin embedding means that epitopes undergo denaturing and even become lost during fixation. Proteintech antibodies are suited to these conditions as they are raised against the full-length protein, giving them an advantage as they naturally recognize more epitopes. This increases the chance of them binding to their target protein both before and after FFPE.
Today’s experiment – staining with anti-LGR5
Today Joanna and her team are testing (along with several others) new polyclonal antibody LGR5 (you can find out more about it’s targeted protein here). Armed with a wealth of information from the Proteintech research team, including literature observed staining patterns, Joanna has selected human normal small intestine and colon cancer sections to test the antibody.
As LGR5 expression has been well-characterized in both these types of tissue I’m satisfied this is a good starting point. We decide to conduct the IHC staining with two dilutions: 1:50 and 1:100 – as well as a negative control (i.e. the primary antibody will be omitted but the secondary antibody will still be applied) for each tissue type. We then get to work deparaffinizing and rehydrating the sections…
We decide to omit the antigen retrieval step as it is not always necessary; this is because tissue loss is generally experienced with this process, even though it can help to achieve more successful staining. I begin to feel that an unfamiliar IHC experiment may involve a little divination when it comes to deciding on things like antigen retrieval. For instance, with this step in particular, it largely depends upon the location of epitopes within the tissue sample – if they’re all sequestered away within the tissue (for instance they might be sandwiched between other non-specific epitopes and the glass of the slide) then antigen retrieval is more likely to be required. It’s just as well most Proteintech antibodies recognize a wealth of specific epitopes then; plus, Joanna reassures me we can revisit this step in another experiment should our staining be unsuccessful this time (as is the standard practice in the lab), but for now it is better to preserve what tissue we have on our slides.
With the decision to bypass antigen retrieval and our samples fully rehydrated we get straight to the immunohistochemical staining. A 3% hydrogen peroxide solution is added to our samples for ten minutes (to suppress any endogenous peroxidase activity which may give false-positive results) in between copious rinsing with Tris buffered saline (TBS). We then choose to block non-specific proteins with 3% bovine serum albumin in TBS for an hour before incubation with our primary antibody anti-LGR5 at the chosen dilutions for another 60 minutes.
Between reagent incubations Joanna gives me a lesson on histology and pathology – using our successful PAX8 antibody (10336-1-AP) as a guide. I’m shown examples of PAX8 staining in the kidney – where I learn the difference between proximal and distal tubules – thyroid and thyroid cancer, and several types of cancerous ovarian tissue.
After the appropriate washing steps either side of an incubation with anti-rabbit, horse radish peroxidise (HRP)-conjugated secondary antibody, we’re ready to develop the color on the slides. We next incubate with the DAB substrate solution that will react with the HRP molecules on any successfully bound secondary antibodies, producing the characteristic brown oxidation product of IHC, easily visible with light microscopy. The typical brown stain appears before our eyes in a matter of minutes. We rinse the stained sections with water before counterstaining with the red-colored compound hematoxylin – this stains the cell nuclei blue.
We then take the necessary steps to fix and mount the tissue so the results can be viewed on a the microscope; this takes around another hour of TBS washing and soaking and transfer to various percentages of ethanol solution, finishing with a final few soaks in xylene. The next step is to seal the work with a coverslip, by which time the suspense has well and truly mounted (an unintentional pun, honest!) Once the sealant is dry, we take the slides over to the microscope and fire up the necessary computer software to record our work for the day.
When the first slide comes into focus I am a little disappointed at the sea of blue nuclei signifying a lot of unstained cells – though I am impressed at how our chosen tissue has retained its micro-structure: the villi of the small intestine are clearly visible. Despite the lack of staining however, it doesn’t take me long to remember we’re not looking for many cells here – LGR5-positive cells should only number a few in each villi and these should be located nearer the bottom of these structures. Looking at the slide it’s clear we have some contenders for specific staining and LGR5-positive cells. Cross referencing our literature sources Joanna and I both agree we might have a specific antibody on our hands here, yet we decide to leave the final decision to a contact of ours and expert in the LGR5 field. We view the other slides and decide to send them to our contact too.
Leaving the department at the end of a long day I thank Joanna for showing me the ropes and leave with deeper understanding of the IHC application and a budding interest in tissue anatomy.
Personally, I would like to continue my exploration of IHC technologies and appeal for any would-be IHC tutors to come forward and teach by example – do you have any IHC results you’d like to talk me through? Maybe you’ve obtained some potential educational material with a Proteintech antibody? If so I’d love to hear about it!*
As to the broader aims of this blog post, I hope it has conveyed the dedication and lengths Proteintech staff go to, to make sure your antibody is validated in - among others - the IHC application. It truly is a company “all about the science”, and strives to be an antibody provider that people feel they can trust – at least that is in my (if somewhat biased) opinion.
*Email any IHC feedback you have to Europe@ptglab.com making sure you quote ‘Immunohistochemistry focus’ as a reference.