Mounting and Coverslipping Mouse Brain Sections
Naveen Ouellette, Holly Myers, daphne.toglia
Abstract
This protocol details mounting thin sliced mouse brain tissue sections onto charged slides in a uniform orientation that does not create wrinkles or artifacts that might interfere with imaging, and then how to apply a coverslip. After mounting and coverslipping, slides will be ready for imaging.
Before start
Mouse brain slices must first be fixed in 4% PFA and stained with desired dyes or antibodies, and typically stored in a well plate prior to mounting. Refer to protocols for sectioning and staining tissue to prepare tissue prior to mounting: Sectioning Mouse Brain with Sliding Microtome and Immunohistochemistry (IHC) Staining Mouse Brain Sections.
Steps
Mounting
Prepare a petri dish filled with 1xPBS & Triton X-100 0.2%.
Place a drop of 1xPBS onto a 1"x3" slide.
Using a small paintbrush, lift the first tissue section to be mounted out of the storage well plate and place section into the petri dish so that it lays flat close to the surface of the 1xPBS & Triton X-100 0.2% solution.
Using paintbrush, lift the tissue section out of the petri dish and place it onto the slide. Arrange tissue section with paintbrush to ensure section is properly aligned, flat, and wrinkle-free with small paintbrush. This can usually be done by eye, but a dissection scope may be used if desired at times when it is difficult to see fine anatomical detail in the slices macroscopically (just by eye).
Repeat steps 2 -4 until all sections have been mounted onto the slides.
Protect slides from light and allow slides to dry at Room temperature for a minimum of 1h 0m 0s or until adhered to the slide.
Once sections are dry, rinse off 1xPBS residue by briefly rinsing the slide with Milli-Q water.
Blot off any residual Milli-Q water by pressing the edge of the slide onto a kimwipe.
Coverslipping
Choose requested mounting medium:
| A | B |
|---|---|
| Vectashield hardset anti-fade mounting medium, Vector Laboratories (H-1400-10) | |
| Vectashield hardset anti-fade mounting medium with dapi, Vector Laboratories (H-1500-10) | Generally not requested if dapi was already used as a secondary stain. Staining with dapi as a secondary stain provides stronger staining than vecta shield with dapi. |
| ProLong Glass Antifade Mountant (P36980) | Good for minimizing optical refraction and preserving fluorescent signal |
Coverslip slide with mounted tissue:
Place a few drops of chosen mounting medium onto the slide to be coverslipped, making sure there are no bubbles. If mounting medium bottle does not have dropper attachment, use a pipette to place drops on slide.
Start with only one edge of the coverslip touching the corresponding edge of the slide, and slowly lay down the coverslip.
If re-coverslipping is required due to air bubbles overlapping onto tissue sections or other artifacts, soak slide in 1xPBS in a petri dish until coverslip comes off easily and repeat step 10. Do not use force to pull off a stuck coverslip; soak the slide longer.
Allow slides to dry at Room temperature, protecting from light by covering slides with a sheet of aluminum foil, a box top, or other material that will shield slides from light.
Store slides at 4°C protected from light until ready for imaging. Slides can be stored for several months.
