Coating (or ‘subbing’) slides for immunohistochemistry can be the difference between having an organized set of tissue slices ready for microscopy- or watching them detach and float away during a wash. It takes a lot of time to place tissue slices in correct anatomical order, aligned right-side up and flat. To the naked eye, all microscope slides may appear identical. Why is ‘subbing’ so important? Unsubbed microscope slides are exactly what you would expect: standard, everyday glass slides.
We’re not staying where we should!
Unfortunately, tissue slices don’t always like to stay put on plain glass slides. An extra molecular foundation is needed to help the tissue stay connected permanently throughout the various staining and washing steps of your experiment. Subbed slides are often pre-treated in a bath containing gelatin and another chemicals such as ‘chrom alum’ so the surface of the slide is very sticky to the surface of the tissue. Some immunohistochemistry labs may purchase pre-cleaned, pre-subbed slides by the case. And that means they’ll rarely ever think about preparing their own in-house. Sometimes only a small batch of slides needs subbing.
Here’s a basic protocol to get started:
- Load a slide rack with glass microscope slides.
- Pre-clean the slides by rinsing twice in a container with deionized water.
- Rinse with 100% ethanol.
- Air or drip-dry, covered, in a low-dust area of the lab.
- Prepare a subbing solution with 0.5 % gelatin in warm deionized water.
- Cool to room temperature.
- Dissolve chromium potassium sulfate (‘chrom alum’) in the solution (0.05 % w/v, or 0.5 g per 1litre solution).
- Immerse slides in a container with subbing solution for about a minute or two.
- Let slides air or drip-dry, covered in a low-dust area of the lab.
- Store slides, covered, indefinitely.
- Properly dispose of subbing solution.
A flattening challenge
Don’t have chrom alum available in the lab? That’s okay – subbing can be done successfully with gelatin alone. If you feel that slices are still not staying where they should be, then try increasing the amount of gelatin dissolved in solution (i.e. 1-3 %) and repeating the sub/dry portion of the protocol (steps 7 & 8) two or three times to make a thicker coating on the slide. One consequence of increasing the gelatin concentration is that it may be more challenging to flatten tissue slices once placed upon the slide.
Not all reagents are gentle to tissue slices, which could be causing them to detach and float away from an unsubbed slide. Chrom alum has an interesting property which makes it a really good additive. In alkaline solutions, molecular bonds between chromium and oxygen are very stable. Oxygen atoms are everywhere throughout the slide- as well as tissue proteins- and chrom alum cements the two together.
The trick is to find a good glue
Subbing can be done with a variety of different chemicals, like poly-d and poly-l-lysine, silanes, collagen and even nitrocellulose films. Whatever your ingredient of choice, the trick is to coat the glass slide with a good chemical ‘glue’ which will hold your tissue slices in place until finishing with cover glass.
Which method do you use to make sure your sections stay where they are meant to?