Mar 25, 2024

Public workspaceDAPI Staining Mouse Brain Sections

DOI
dx.doi.org/10.17504/protocols.io.3byl4jm6rlo5/v1
  • 1Allen Institute for Neural Dynamics;
  • 2Boston University
daphne.toglia
Open access
DOI: dx.doi.org/10.17504/protocols.io.3byl4jm6rlo5/v1
Protocol Citation: daphne.toglia, Holly Myers 2024. DAPI Staining Mouse Brain Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4jm6rlo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 01, 2023
Last Modified: June 18, 2024
Protocol Integer ID: 82765
Keywords: DAPI staining, Histology
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.
Materials
ReagentDAPI Thermo Fisher ScientificCatalog #D1306 (Concentration5 mg/mL )
ReagentPhosphate Buffered SalineThermo Fisher ScientificCatalog #28374
ReagentSodium azideP212121

MaterialsProduct number
Aluminum foilAmazon, B074NB5CDZ
48 well plate or equivalent staining plateCostar, 3548
Corning 50ml centrifuge tubeMillipore Sigma, CLS430829
15ml centrifuge tubeGlobe Scientific, 6264
Benchmark vortex mixerSouthern Labware, BV1000
Manual single channel pipettes: P20, P200, P1000Rainin, 30456871
Manual single channel pipette: P2Rainin, 17014393
Manual single channel pipettes: P5000Rainin, 17011790
Nutating mixerFisherbrand, 88-861-043
Stir barGrainger, 21R590


1L 1xPBS:

Combine the following reagents into a container with a stir bar. A graduated cylinder may be used to measure MilliQ water and a graduated cylinder or serological pipet may be used to measure 10xPBS. Mix well on a stir plate at high speed (300 RPM or higher) for Duration00:02:00 or until solution is mixed. Store at TemperatureRoom temperature for 1 month.
ReagentVolume
Milli-Q water900mL
10xPBS100mL

1L 1xPBS & 0.01% Sodium Azide:

Combine the following reagents into a container with a stir bar, using a graduated cylinder to measure the 1xPBS and a P5000 pipette to measure the 0.01% sodium azide. Mix well on a stir plate at high speed (300 RPM or higher) for Duration00:02:00 or until solution is mixed. Store at TemperatureRoom temperature or Temperature4 °C for up to 1 year.


ReagentVolume
1xPBS999mL
Sodium Azide1mL

2mL 5mg/mL DAPI solution:

Add Milli-Q water to DAPI powder in 10mg vial using P5000 pipette. Vortex until powder completely mixes into solution. Store at Temperature4 °C and vortex before use.

ReagentVolume
Milli-Q water2mL
DAPI powder10mg

Safety warnings
Attention
DAPI is a mutagen and should be handled with care. Wear PPE and dispose into hazardous waste stream. Please consult your immediate supervisor or the EH&S manager/representative if you have questions or concerns.

Sodium Azide is toxic and carcinogenic. It should be handled and prepared with care. Do not breathe dust, do not use metal utensils. Wear gloves when handling this chemical.

Paraformaldehyde is carcinogenic. Wear gloves at all times when handling specimen fixed in paraformaldehyde, as the tissue may contain trace amounts of the chemical.
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.


Before Staining
Before Staining
Wash the mouse brain sections for at least 3 times, Duration00:05:00 each time in 1XPBS.


Safety information
Paraformaldehyde is carcinogenic. Wear gloves at all times when handling specimen fixed in paraformaldehyde, as the tissue may contain trace amounts of the chemical.

5m
Diluting DAPI
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.


Safety information
DAPI is a mutagen and should be handled with care. Wear PPE and dispose into hazardous waste stream. Please consult your immediate supervisor or the EH&S manager/representative if you have questions or concerns.


Note
Volume will vary based on the amount of solution needed. For a standard 48 well plate, 200uL of DAPI solution should be prepared for each well in order to fully submerge the tissue in solution, and this may be used to calculate the total volume of solution needed.

If staining in a plate with larger wells (ex: 24 well plate, 12 well plate, or Netwell cell culture inserts), a larger volume of DAPI solution will be needed in order to fully submerge the tissue in solution, and this should be used to calculate the total volume of solution needed.

Example recipe for 5mL total volume DAPI:1xPBS solution:
ReagentVolume
1xPBS5mL
DAPI1uL



Staining with DAPI
Staining with DAPI
50m
Pipette diluted DAPI (see note below) into each well with a manual pipette and place well plate on a shaker at TemperatureRoom 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.

Section thicknessStaining duration
50 microns15 minutes
100 microns30 minutes


Safety information
Sodium Azide is toxic and carcinogenic. It should be handled and prepared with care. Do not breathe dust, do not use metal utensils. Wear gloves when handling this chemical.


Note
200uL is sufficient volume to full submerge tissue in diluted DAPI solution in each well in a standard 48 well plate. If staining in plates with larger wells (ex: 24 plate well, 12 plate well), a larger volume of solution may be needed to fully submerge the tissue in each well.

After the incubation period, wash sections at least 5 times at Duration00:05:00 each with 1xPBS. Refer to protocol Mounting and Coverslipping Mouse Brain Sections for next steps.
25m
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 Temperature4 °C until ready for mounting.