My staining didn’t work, part 3: IHC and IF staining tips

It can be frustrating to get to the end of an immunohistochemistry (IHC) or immunofluorescence (IF) experiment just to find out the staining didn’t work. While there are some standard steps to take to solve problems you might come across, sometimes you need to look outside the box. Here we discuss some miscellaneous factors to focus on when an IHC or IF protocol performs unexpectedly. 

This blog post is the third of a three-part series intended to help researchers navigate different parts of the IHC and IF staining processes that might present challenges. You can check out the first part of the series, “Background staining in IHC and IF“, and the second the second part of the series, “Absence of positive staining in IHC and IF“. 


With modern techniques, antibodies can be raised in a variety of species: mouse, rat, goat, horse, and more. Species choice is important to minimize cross-reactivity, but sometimes it is inevitable to work with species-on-species reagents and tissues. For example, it is problematic to use a mouse primary antibody on mouse tissue because the anti-mouse secondary antibody will recognize endogenous mouse immunoglobulins in addition to the primary antibody (1,2). Certain kits, such as Vector M.O.M.® (Mouse on Mouse) Immunodetection Kits will block endogenous mouse immunoglobulins, making it possible to fluorescently detect one or more mouse primary antibodies on mouse tissue.  

Unwanted species interaction can also occur with xenograft models, for example the study of a human tumor grown in a mouse model (3). While the tissue itself is human derived, it will be vascularized with mouse blood vessels and contain mouse immunoglobulins. Applying an anti-mouse secondary antibody may produce cross-reactivity with the xenograft tissue, leading to potential false positives. In this situation, similar M.O.M. reagents can ameliorate the issue. Anti-rat and anti-hamster secondary antibodies also have a degree of cross-reactivity with mouse tissue because they are related rodent species. To find out how to eliminate unwanted binding of a closely related species, check out our first blog post from this series. 


Counterstaining IHC/IF samples spatially orients the target within its surroundings, which can help guide researchers as they analyze their results. Ensure judicious use of a counterstain, as heavy staining may mask weakly expressed antigens. Additionally, too similar of a substrate color can obscure target antigens causing false positives or false negatives. To navigate these challenges, make sure the counterstain and initial detection systems provide adequate contrast and have compatible protocol parameters such as solubility and heat resistance, if applicable. Researchers should run counterstain-only controls to validate their chosen counterstain performance. Running a titer can help determine the best counterstain protocol prior to proceeding with the antigen targets.  


IHC and IF protocols can be adapted to detect more than one target in a sample, allowing detailed analysis of different targets in parallel. With these more advanced experiments, keep in mind some not-so-obvious steps which may negatively affect your stain if not addressed.  

Watch out for potential incompatibilities between secondary antibodies. While it is usually no problem to use two secondary antibodies raised in the same species, issues may arise with some antibody combinations. For example, a secondary antibody raised in horse in the initial labeling reagents and an anti-horse secondary in the subsequent labeling reagents would lead to cross reactivity. To address this without changing antibodies, run the staining workflow sequentially through to the initial system development prior to the subsequent labeling system. 

In an avidin/biotin detection system, it can be necessary to block endogenous avidin and biotin to mitigate background noise (4). This is typically done before the first primary antibody incubation, but when detecting multiple antigens, it is necessary to block before each primary antibody to prevent the subsequent set of labeling reagents from interacting with the prior set of labeling reagents.  

Once the detection systems are determined, it is best to optimize single labeling protocols prior to combining them into a multiplex protocol (1). This optimization has a second benefit: the stained sections from the single label protocols can be used as controls to compare the multiplex staining quality. 

Microscopy setup 

IHC slides are typically viewed under a bright-field microscope. There are some parameters to keep in mind, like the brightness adjustments and condenser, but there are more specific parameters to check off with fluorescent microscopy in IF. It is necessary to define the proper excitation and emission spectrum for targets. Each fluorophore emission spectra should ideally have a narrow peak with minimal overlap to optimize each signal distinctly (5). Ensure the appropriate filters are chosen to maximize the target fluorophore emission intensity while minimizing the unwanted signal from other fluorophores. 


Sometimes even the miscellaneous category has a miscellaneous category. Here are some extra steps to review when assessing why the staining did not work.  

IHC and IF kits are widely available and offer a convenient number of compatible products for a protocol, but not all kits cover every step. Ensure every step from beginning to end is accounted for, including all the necessary ancillary components, and confirm compatibility throughout the full workflow. 

Each product will have specific storage conditions and expiry dates. Using a product outside of these specifications can negatively impact the stain results. Take care to store all reagents as recommended and use them within the denoted expiry date. Key factors to keep in mind are storage temperature, protection from light, and reduction of freeze-thaw cycles. Additionally, while a product concentrate may be stable for many months, the working solution may need to be made fresh prior to each use. 

While this is not an exhaustive list for why a staining did not turn out as expected, it contains great clues for the detective’s journey to solving the case. For more tips and tricks to improve your staining, check out our IHC and IF Resources, and stay tuned for more help on the blog. 


  1. Van der Loos, CM. 2007. Multiple Immunoenzyme Staining: Methods and Visualizations for the Observation With Spectral Imaging. Journal of Histochemistry & Cytochemistry.  
  2. Goodpaster T, et al. 2013. A Flexible Mouse-On-Mouse Immunohistochemical Staining Technique Adaptable to Biotin-Free Reagents, Immunofluorescence, and Multiple Antibody Staining. Journal of Histochemistry & Cytochemistry.  
  3. Cekanova M, et al. 2014. Animal Models and Therapeutic Molecular Targets of Cancer: Utility and Limitations. Drug Design, Development and Therapy 
  4. Bussolati G, et al. 2003. Retrieved Endogenous Biotin: A Novel Marker and a Potential Pitfall in Diagnostic Immunohistochemistry.  
  5. Evans K, et al. 2013. Chapter 9.11 – Hematology. The Immunoassay Handbook (Fourth Edition).