Special stains are my favorite. So many vibrant colors to highlight specific tissue components. Magenta for glycogen, blue for collagen and mucin, red for muscle fibers and cytoplasm, and many more! 

Special stains look like pieces of art and are used when a routine H&E stain does not show quite enough for a pathologist to see what’s going on. They can be used to identify tissues and structures and complement other tests to detect pathogens or diagnose cancer. With many different stains and options and so many colors and chemical reactions taking place, there is nothing to be afraid of when doing these stains manually. Knowing some simple tips and tricks to navigate through special stains will help to ensure successful results. 

1. Is a protocol provided with the kit? Follow it! Protocols are optimized to work best with specific reagents and concentrations provided in the kit. Skipping or modifying steps can alter the results of the stain. Following the protocol also ensures consistency each time the stain is performed. 
2. Rinse times are critical! Don’t try to save time by eliminating or reducing rinse steps. Rinses are critical to completely remove reagent to not interfere with the next step. Whenever possible, use DI water since tap water may contain microorganisms that could cause a false positive.  
3. How clean is your glassware? Some reactions, such as those with silver, require chemically clean glassware or excess precipitate can occur that may interfere with a diagnosis. Don’t let this scare you though! Cleaning glassware can be as simple as rinsing immediately after use, letting soak in a 1% bleach solution for 20 minutes, and 5 thorough rinses in running tap water followed by 3 thorough rinses in DI water. That’s it! 
4. Is a working solution required? Don’t prepare it too far in advance. Most working solutions will start to break down in about an hour and this can alter the reaction. For optimal performance, prepare working solutions just before completing the stain. Working solutions are typically intended for single use and should not be reused. 
5. Get everything ready before you start. Are there multiple steps with many reagents and supplies? Don’t be overwhelmed! Gather all supplies and reagents needed and set them up in the correct order. Then all you need to do is move from one step to the next following the protocol. 
6. Are any reagents stored in the refrigerator? Allow time for them to come to room temperature before use. If not at room temperature, the reagent may “shock” the slide and result in incorrect staining results. 
7. Did you know that counterstain times can be modified? Counterstains provide a visual contrast to the area of interest and in most cases can be modified to the desired preference. In some instances, a range of time is listed for the counterstain. Document the preferred time or any changes in times for consistent results. 
8. The recommended protocols are generally for FFPE human tissues. Are you in a research lab or working with animal tissues? You can still use these protocols as a starting point. Stain a test slide or two using the provided protocol and adjust from there. In many cases, animal tissue will stain much lighter. If so, try doubling the times and see if that achieves your lab preference. Follow the validation protocol according to your lab SOPs and document any changes made.

Remember that these are bits of helpful advice to assist with your special stains journey. Every special stain is unique and may have its own challenges to learn how to tackle, but there are sometimes steps that can be reversed or repeated. Have questions or need some assistance? Contact our Product Support Team to help!