Handling Free-Floating Sections
Recommended Supplies
- “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)
Section Preparation
- 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
- Wet the slide with mounting solution before placing the section onto the slide.
- Swirl the solution in the dish with the brush to elevate the section from the bottom.
- Alternately move the corners of the section up the “ramp” of the slide, continuing to scoot the section to it’s “final destination”. The gelatin matrix is sturdy, thus make efforts during adjustments to contact the gelatin rather than the tissue portion of the Multi-Brain section. Smooth gentle motions will ensure success.
- Gradually withdraw and elevate the slide from the mounting solution. Tip the slide to promote more drainage, while adjusting the section to its proper position. Make fine adjustments as needed to evenly distribute the section onto the slide. Pockets of fluid may create the appearance of a pucker, draining will flatten the section.
- Depending on the stain applied to the sections, the ideal pH of the mounting solution will vary. Sections that are mounted prior to staining (e.g. Nissl, H&E,) normally do best beginning with a 4.9 pH buffered mounting solution. Sections that are mounted after staining (e.g. IHC and some specialty stains) normally do best beginning with a 6.0 pH buffered mounting solution. For fine adjustments to the pH see below:
- If the brain tissue appears too big to “fit” into the gelatin matrix adjust the pH LOWER using 1M acetic acid (e.g. if allowed to dry, the section would show many overlapping wrinkles).
- If the section appears to be pulling the gelatin inward adjust the pH HIGHER using 1M ammonium acetate (e.g. if allowed to dry, the gelatin would show puckering).
| Stock Acetate Buffers: | ||
| For reference: | 1M Acetic Acid (15mL Glacial Acetic Acid/ 235 mL H2O) | |
| 1M Ammonium Acetate (19.27g/250 mL H2O) | ||
| Working Acetate Solution: | ||
| 4.9 pH = | 15 mL (1M) 35 mL (1M) | Acetic Acid Ammonium Acetate |
| 6.0 pH = | 1.875 mL (1M) 48.125 mL (1M) | Acetic Acid Ammonium Acetate |
| Subbing Solution: | ||
| 0.1g | Gelatin | |
| 20 mL | H2O @ 60° C | |
| Stir until dissolved | ||
| Working Solution: | ||
| 188 mL | dH2O | |
| 12 mL | Acetate Buffer (4.9 or 6.0 pH) | |
| 50 mL | 95% ETOH | |
| 2 mL | Subbing Solution |
Mounting Instructional Video:
https://youtu.be/KEpyRzCtrKk&rel=0
For more information:
