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

Take a chemically fixed mouse brain slice embedded in a tissue-embedding medium.

Wash the slice with buffer to remove the medium.

Add a solution that permeabilizes the membranes and blocks non-specific binding sites.

Remove the solution.

To visualize neuronal synaptic proteins, incubate the slice with primary antibodies.

These antibodies target a presynaptic protein expressed in the synapses of specific brain regions and a ubiquitously expressed synaptic protein as a reference marker.

Wash with buffer to remove unbound antibodies.

Introduce fluorophore-labeled secondary antibodies that target the primary antibodies.

Wash with buffer to remove unbound secondary antibodies.

Counterstain the nuclei with a DNA-binding dye.

Wash with buffer and mount the slice using a suitable mounting medium.

Visualize the slice under a confocal microscope.

Calculate the mean fluorescence intensity ratios of the target protein and the reference marker to analyze the target protein's distribution across different brain regions.

To prepare the slices for immunostaining, use a plastic pipette to remove the PB solution from one well without drawing in the brain slices. Then, use a 1,000-microliter pipette to add 250 microliters of fresh PB to wash them of excess OCT. Repeat this washing for each well at the time to avoid drying out the slices.

Then, use a plastic pipette to remove the PB solution from the first well. Use a 1,000-microliter pipette to add 250 microliters of blocking buffer per well, working well by well again. Incubate the plate at room temperature on a shaker for three hours. During the incubation at 250 microliters of antibody buffer per well to a reaction tube.

Then, use a 2-microliter pipette to add the appropriate amount of antibody, pipetting it directly into the solution, and gently pipette up and down several times to mix. Then, vortex this diluted antibody to ensure proper mixing.

Working well by well, remove the blocking buffer with a plastic pipette and add 250 microliters of primary antibody solution per well. Incubate the plate on a shaker at 4 degrees Celsius overnight.

The following day, remove the antibody solution with a plastic pipette. Wash the slices with 300 microliters of washing buffer 1 per well, three times 10 minutes each wash on a shaker at room temperature. During the washing, working in the dark, dilute the fluorophore-coupled secondary antibody in a reaction tube in the same fashion as previously done with the primary antibody.

After completed washing, remove the washing buffer with a plastic pipette and add 250 microliters of secondary antibody solution per well. Incubate in the dark at room temperature for 90 minutes. After completed incubation, remove the antibody solution with a plastic pipette. Wash the sections three times with washing buffer 2 in the same way as with washing buffer 1.

During this washing, dilute DAPI stain in 0.1 molar PB to achieve a 1-to-2000 concentration. After removing the washing buffer from the plate, add 250 microliters of DAPI solution, and incubate at room temperature on the shaker for 5 minutes.

After removing the DAPI solution with a plastic pipette, use a 1,000-microliter pipette to add 500 microliters of 0.1 molar PB per well. Place a microscope slide under a stereoscope. Use a fine brush to add three separate drops of 0.1 molar PB onto the slide. Using the brush, place one slice per drop on the slide, and then, flatten and orient the slices.

After all slices are positioned correctly, use a paper tissue to remove excess PB and dry carefully without drying the slices completely. Then, add 80 microliters of embedding medium onto the slide, and carefully cover it with a coverslip to embed the brain slices.

Cover the slides to avoid light exposure and leave them to dry in the fume hood for one to two hours, and then store them in a microscope slide box at 4 degrees Celsius until ready for confocal microscopy.

After acquiring virtual tissues of the whole brain slice for different channels as described in the manuscript, load all single channels for one image into Fiji by clicking File and then Open. Then, use the freehand selection tool to delineate one hemisphere in the DAPI channel. Click on Edit, then Selection, and then create mask to create a mask of the selected region.

Then click on Analyze, and then Measure Particles to determine the mean fluorescence intensity for single channels, making sure to select different channels to determine the mean fluorescence intensity values for each channel.

After that, copy the mean fluorescence intensity for the single channels into a spreadsheet. To determine the mean fluorescence intensity for the single channels in an area of interest, delineate the area with the freehand selection tool.