DAPI Staining Mouse Brain Sections
Holly Myers, daphne.toglia
Abstract
This protocol details the steps for staining 4% PFA fixed mouse brain tissue sections with DAPI (4',6-diamidino-2-phenylindole), a fluorescent stain that can be used to label anatomic regions of interest in a mouse brain, allowing for imaging with a fluorescent microscope. The protocol includes a table with suggested staining duration based on section thickness and guidelines for calculating the volume of DAPI solution needed for staining.
Before start
This protocol details DAPI staining for 4% PFA fixed mouse brain tissue that has already been sliced. Refer to protocol Sectioning Mouse Brain with Sliding Microtome for information about preparing slices ahead of DAPI staining.
Throughout this protocol, tissue should be protected from light by covering well plate with aluminum foil between steps or equivalent protective measure.
Steps
Before Staining
Wash the mouse brain sections for at least 3 times, 0h 5m 0s each time in 1XPBS.
Diluting DAPI
Dilute 5mg/mL DAPI solution to 1:5000 with 1xPBS by measuring 1XPBS into a conical tube and adding DAPI with a manual pipette. Vortex DAPI before pipetting into 1XPBS.
Staining with DAPI
Pipette diluted DAPI (see note below) into each well with a manual pipette and place well plate on a shaker at Room temperature. Each well should contain enough DAPI solution to fully submerge the sections within. Leave well plate on shaker for suggested staining duration based on section thickness (see table below). Ensure that the well plate is covered in aluminum foil and away from direct light.
| A | B |
|---|---|
| 50 microns | 15 minutes |
| 100 microns | 30 minutes |
After the incubation period, wash sections at least 5 times at 0h 5m 0s each with 1xPBS. Refer to protocol Mounting and Coverslipping Mouse Brain Sections for next steps.
If unable to mount on the same day:
After 1X PBS washes, place sections in 1xPBS & 0.01% sodium azide solution, cover well plate with aluminum foil to protect from light, and store in 4°C until ready for mounting.
