Quantifying the Distribution of a Presynaptic Protein in the Mouse Brain Using Immunofluorescence

Protocolo

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Immunofluorescence

1. Prepare solutions including the blocking buffer, antibody buffer, washing buffer 1, and washing buffer 2 (see Table 1).
2. Rinse slices once with phosphate buffer (PB) to remove excess optimal cutting temperature (OCT) compound.
   1. Remove the solution with a plastic pipette without sucking in the brain slices. Add 250 µL of fresh PB with a 1000 µL pipette.
      CAUTION: Slices should not dry out, so remove and add fluids well by well.
3. Remove the PB with a plastic pipette and add 250 µL of blocking buffer per well with a 1000 µL pipette. Incubate for 3 h at room temperature (RT) on the shaker.
4. During the incubation time, dilute the primary antibodies in antibody buffer in a reaction tube. Use 250 µL antibody buffer per well and add the appropriate amount of antibody (see Table 2) by pipetting it directly into the solution using a 2 µL pipette. Mix the solution by gently pipetting up and down several times. Vortex shortly afterwards to ensure proper mixing.
   NOTE: To determine the background fluorescence, staining should also be performed without adding the primary antibody. For that, incubate the slice in antibody solution without primary antibodies according to the protocol.
5. After the incubation time, remove the blocking buffer with a plastic pipette and add 250 µL of antibody solution containing primary antibodies per well. Incubate slices with primary antibody overnight at 4 °C on a shaker.
6. Next day, wash the slices with washing buffer 1 3x for 10 min at RT on a shaker.
   1. Remove the medium with a plastic pipette and add 300 µL of washing buffer 1 per well. Incubate at RT for 10 min. Repeat 3 times.
7. During the washing steps, dilute the fluorophore-coupled secondary antibodies in antibody buffer in a reaction tube. Use 250 µL antibody buffer per well and add the appropriate amount of antibody (see Table 2) by pipetting it directly into the solution using a 2 µL pipette. Mix the solution by gently pipetting up and down several times. Vortex shortly afterwards to ensure proper mixing.
   CAUTION: Because the antibodies are light-sensitive, all steps from this point on need to be performed in the dark.
8. After the washing steps, remove the washing buffer with a plastic pipette and add 250 µL of antibody solution containing secondary antibodies per well. Incubate the slices with secondary antibody for 90 min at RT in the dark.
9. Wash the slices with washing buffer 2 3x for 10 min at RT.
10. During the washing steps, dilute 4′,6-diamidino-2-phenylindole (DAPI) in 0.1 M PB in a concentration of 1:2000.
11. Remove the washing buffer 2 with a plastic pipette and add 250 µL of DAPI solution per well. Incubate for 5 min at RT on the shaker.
12. Remove the DAPI solution with a plastic pipette and add 500 µL of 0.1 M PB per well with a 1000 µL pipette.
13. Mount slices on microscope slides.
    1. Place a microscope slide under the stereoscope. With a fine brush, add three separate drops of 0.1 M PB onto the slide. Place one slice per drop onto the microscope slide.
    2. Use the fine brush to flatten and orient the slices on the microscope slide.
    3. When all slices are positioned correctly, remove excess PB with a tissue and dry the slide carefully.
       CAUTION: Avoid drying the brain slices completely.
    4. Add 80 µL of embedding medium onto the slide. Carefully lower the coverslip onto the slide, thereby embedding the brain slices.
    5. Leave the slides to dry in the fume hood for 1-2 h (cover them to avoid light exposure) and store them in a microscope slide box at 4 °C.
       NOTE: The protocol can be paused here.

2. Imaging

1. After the embedding medium is completely hardened, place the microscope slide under the confocal microscope.
   NOTE: Epifluorescence microscopy combined with deconvolution software should yield similar image quality.
2. Adjust the laser settings by increasing or decreasing the laser intensity for every channel so that few pixels are overexposed to ensure maximum distribution of grey values.
3. Acquire virtual tissues of the whole brain slice for the different channels.
   1. In the imaging software, select the Tiles option and manually delineate the brain slice with the Tile Region Setup.
   2. Distribute support points throughout the tile region and adjust the focus for the different support points by pressing Verify Tile Regions/Positions….
   3. Adjust the settings in Acquisition Mode according to the desired resolution and file size of the resulting image and start the scan.
4. When the scan is finished, use the Stitching function to process the virtual tissue. Export the file as a .tif with the function Image Export.

3. Computer-based Analysis

1. Load all single channels for one image into FIJI by clicking File| Open.
2. With the Freehand selection tool, delineate one hemisphere in the DAPI-channel. Create a mask of the selection by clicking Edit| Selection| Create mask.
3. Determine the mean fluorescence intensity for the single channels (Mover and synaptophysin) by clicking Analyze| Measure.
   NOTE: Make sure to select the different channels to determine the mean fluorescence intensity values for each channel.
4. Copy the mean fluorescence intensity for the single channels into a spreadsheet.
5. Determine the mean fluorescence intensity for the single channels in an area of interest by delineating the area also with the Freehand selection tool. Use a mouse brain atlas as reference.
6. Repeat steps 3.1-3.5 for all hemispheres and all areas of interest.
   NOTE: Determine the values for each hemisphere separately in order to later compare the values in an area of interest to that in the hemisphere (see step 4.2).

4. Data Handling

1. In case the background fluorescence is high, a background subtraction might be needed. For that, determine the mean fluorescence intensity for the slice processed without primary antibody against the reference protein (here: synaptophysin) and subtract that value from all values obtained for the brain regions and hemispheres.
2. When the mean fluorescence intensities for the single channels for every hemisphere and every area of interest have been determined (see Table 3), calculate the ratio of Mover to synaptophysin by dividing the value for Mover by the value for synaptophysin (yellow in Table 3). Perform this action for every hemisphere and every area of interest separately.
3. Divide the ratio obtained for one area of interest by the ratio obtained for the corresponding hemisphere (orange in Table 3) to determine the ratio of the area of interest to the hemisphere.
4. To determine the relative Mover abundance, translate the ratio obtained in 4.2 into a percentage by determining its deviation from 1 (red in Table 3). A ratio of 1.25 would therefore give a relative Mover abundance of 25% above average, and a ratio of 0.75 would yield a relative Mover abundance of 25% below average.

Table 1: Solutions used in this protocol.

Fixative (500mL)
Mix 20g paraformaldehyde (total conc.: 4%) 50 mL 10x PBS stock solution (total conc.: 1x) 450 mL bidest H2OAdjust pH to 7.4 with NaOH Note: To solve the paraformaldehyde in PBS, heat the solution. Do not heat over 70 oC, as PFA disintegrates at temperatures higher than 70 oC. Caution: PFA is toxic, potentially carcinogenic and teratogenic. Wear gloves when working with PFA and work under the fume hood. Avoid ingestion.
0.1M PB (1L)
Stock solution X 35.61 g Na2HPO4.2H2O in 1 L bidest H2O	Stock solution Y 27.60 g NaH2PO4.2H2O in 1 L bidest H2O
Mix 385 mL stock solution X 115 mL stock solution Y 500 mL bidest H2O
Blocking buffer (50 mL)
Mix 1.25 mL normal goat serum (total conc.: 2.5%) 1.25 mL normal donkey serum (total conc.: 2.5%) 0.5 mL non ionic surfactant (Triton-X100, total conc.: 1%) 47 mL 0.1 M PB
Antibody buffer (50 mL)
Mix 0.25 mL normal goat serum (total conc.: 0.5%) 0.25 mL normal donkey serum (total conc.: 0.5%) 0.1 mL non ionic surfactant (total conc.: 0.2%) 49.4 mL 0.1 M PB
Washing buffer 1 (50 mL)
Mix 1 mL normal goat serum (total conc.: 2 %) 49 mL 0.1 M PB
Washing buffer 2 (50 mL)
Mix 0.5 mL normal goat serum (total conc.: 1 %) 49.5 mL 0.1 M PB

Table 2: Antibodies used in this protocol.

Primary antibodies
Directed against	Host species	RRID	Concentration
Mover	Rabbit	AB_10804285	1:1000
Synaptophysin	Guinea pig	AB_1210382	1:1000
Secondary antibodies
Target species	Host species	Fluorophore	Concentration
Rabbit	Donkey	AlexaFluor 647	1:1000
Guinea pig	Goat	AlexaFluor 488	1:1000

Table 3: Example of data handling.

Hemisphere
Hemisphere #	Mean fluorescence intensity Synaptophysin (A.U.)	Mean fluorescence intensity Mover (A.U.)	Ratio Mover/Synaptophysin
1 2 3 4 5 6	29.134 31.008 38.641 30.775 21.658 27.277	22.810 24.046 29.324 25.444 18.091 23.364	=C3/B3      0.783 =C4/B4      0.775 =C5/B5      0.759 =C6/B6      0.827 =C7/B7      0.835 =C8/B8      0.857
Hippocampus
Hemisphere #	Mean fluorescence intensity Synaptophysin (A.U.)	Mean fluorescence intensity Mover (A.U.)	Ratio Mover/Synaptophysin	Ratio to hemisphere	Relative Mover abundance (%)
1 2 3 4 5 6	35.26 33.955 41.231 39.853 30.129 28.737	29.889 27.825 31.978 31.787 27.817 25.861	=C11/B11         0.848 =C12/B12         0.819 =C13/B13         0.776 =C14/B14         0.798 =C15/B15         0.923 =C16/B16         0.900	=D11/D3            1.083 =D12/D4            1.057 =D13/D5            1.022 =D14/D6            0.965 =D15/D7            1.105 =D16/D8            1.051	=(E11-1)*100      8.269  =(E12-1)*100      5.673 =(E13-1)*100      2.200 =(E14-1)*100     -3.528 =(E15-1)*100    10.530 =(E16-1)*100      5.064

Materiales

Name	Company	Catalog Number	Comments
1.5 mL reaction tubes	Eppendorf	30120094
50 mL reaction tubes	Greiner Bio-One	227261
multiwell 24 well	Fisher Scientific	087721H
plastic pipette (disposable)	Sarstedt	8,61,176
1000 mL pipette	Rainin	17014382
2 ml pipette	Eppendorf	3123000012
Vortex Genius 3	IKA	3340001
Menzel microscope slides	Fisher Scientific	10144633CF
Stereoscope	Leica
LSM800	Zeiss		Confocal microscope
PBS (10X)	Roche	11666789001
Tissue Tek	Sakura	4583	OCT
Na2HPO4	BioFroxx	5155KG001
NaH2PO4	Merck	1,06,34,60,500
normal goat serum	Merck Millipore	S26-100ML
normal donkey serum	Merck	S30-100ML
Triton X-100	Merck	1,08,60,31,000
rabbit anti-Mover	Synaptic Systems	RRID: AB_10804285
guinea pig anti-Synaptophysin	Synaptic Systems	RRID: AB_1210382
donkey anti-rabbit AF647	Jackson ImmunoResearch	RRID: AB_2492288
goat anti-mouse AF488	Jackson ImmunoResearch	RRID: AB_2337438
Mowiol4-88	Calbiochem	475904
ZEN2 blue software	Zeiss		Microscopy software
FIJI	ImageJ		Analysis software
Microsoft Excel	Microsoft