Handling Free-Floating Sections
- “Hockey sticks” to transfer tissue (available through our Histotools® store)
- Subbed slides, see Subbing Solution below (both subbed and unsubbed slides are available through our Histotools® store)
- Small glass dish (small casserole dish)
- Small paint brushes (we use Loew Cornell round brushes #1, 2 and 3)
- Acetate (NOT phosphate!) buffer with a pH of ~5 (recipes below)
- Kimwipes (or similar lint-free towel)
- Drying rack (means of propping up slides nearly vertically for draining and drying)
- All MultiBrain®, MultiCord®, and Large Format™ sections are stored in an Antigen Preserve solution, which is especially viscous at ≤ -15ºC. Let the solution warm up to near room temp, then immerse the container in a much larger container (e.g. small casserole dish) that holds deionized water.
- With the entire container submersed, tilt the section container over, keeping it immersed, and allow the sections to drift out. Proceed to transfer the sections to the containers or dishes more appropriately sized for your processing.
Some stains require that the sections be mounted onto slides prior to staining, e.g. H&E and Nissl stains. Most other stains are performed while the sections are “free floating”, e.g. immunohistochemistry stains. These sections are mounted onto slides AFTER the staining.
Mounting Free-Floating Sections
See Video Below
The Antigen Preserve solution is viscous, and even more so when coming straight out of -20ºC. Let the solution warm up to near room temp, and even then it might be less damaging to the tissues (less risk of tearing) to immerse the container in a much larger container holding deionized water. With the entire container submersed, tilt the section container over while immersed and allow the sections to drift out. Proceed to transfer the sections to the containers or dishes more appropriately sized for your processing.
Some stains performed require that the sections be mounted onto slides previous to staining, e.g. Nissl stains. Some other stains we perform must be performed while the sections are “free floating”, e.g. immunohistochemistry stains. These sections are mounted onto slides AFTER the staining.
- With only one or two sections in the small casserole dish, orient one so that it’s just as it will appear on the slide. Leave the section near the middle of the dish. Although the gelatin and tissue are durable, avoid sudden jerking, yanking or strong poking of the sections. Smooth gentle motions will ensure success.
- Wet the slide with the mounting solution before you begin to place the section on the slide.
- Lower the slide at a shallow angle near the edge of the section to be mounted. To elevate the edge of the section nearest the slide, use a gentle swirling action with the brush. By alternately moving the corners of the section nearest the slide up the ‘ramp’ of the slide, continue to scoot the section to the upper part of the immersed slide-it’s final destination. Holding the brush against the upper edge of the section, gradually withdraw and elevate the slide from the mounting solution. If needed, re-immerse to make major adjustments. With the slide now totally out of the solution, tip the slide to promote more drainage, all the time adjusting the section to be in its proper position. Touching the lower corners of the slide to the edge of the mounting tissue will aid in draining off excess solution. Make fine adjustments as needed to get the section evenly distributed on the slide. Some pockets of fluid may create the appearance of a pucker, but draining them will flatten the section.
- Depending on what stain was applied to the sections, the pH of the mounting solution may need to be adjusted. For stained sections that will be mounted first and then stained for Nissl, H&E, myelin etc. start with the 4.9pH buffered mounting solution. For all other sections— mostly those that have been stained with antibodies—start with the 6.0pH buffered mounting solution.
• If the brain sections within the MultiBrain® sheet appear to be too big to fit in the gelatin (if allowed to dry, the brain sections would have many wrinkles), the pH needs to be reduced. This can be accomplished by slightly decreasing the pH by adding a few mLs of the 1M Acetic acid and determining if that is sufficient, and if not, adding more. If there is a really large effect of the brain sections looking as if they are too big to fit in the gelatin, then a bigger ‘jump’ in pH is needed. That is, if you started with the 6.0 pH solution, switch the sections to the 4.9pH.
• If the brain sections appear to be ‘pulling’ on the gelatin, causing ‘puckers’ in the gelatin, then the pH needs to be increased. Small changes can be accomplished by adding a few mLs of the 1M Ammonium Acetate, in a few successions. If the puckering is great, then a bigger jump in pH is needed; i.e. switch the sections to the 6.0pH solution.
|Recipes for Mounting Solutions|
|Stock Acetate Buffers:|
|For reference:||1M Acetic Acid (15mL Glacial Acetic Acid/235 mL H2O)|
|1M Ammonium Acetate (35g/250 mL H2O)|
|Working Acetate Solution|
|4.9 pH = ||15 mL (1M) |
35 mL (1M)
|6.0 pH =||1.875 mL (1M)|
48.125 mL (1M)
|Subbing Solution:||(0.5% Gelatin)|
|20 mL||H2O @ 60° C|
|Stir until dissolved|
|188 mL||d H2O|
|12 mL||Acetate Buffer (4.9 or 6.0 pH)|
|50 mL||95% ETOH|
|2 mL||Subbing Solution|
Mounting Instructional Video: