Here we describe a protocol employing immunofluorescence, confocal microscopy, and computer based analysis to determine the distribution of synaptic protein relative to a reference protein. Our method circumvents the inherent variability of immunofluorescence stainings by using a ratio rather than absolute fluorescence levels. Additionally with this method you can analyze the heterogeneity of protein distribution on different levels.
From whole brain slices to brain regions to even sub regions such as the different layers of the hippocampus. This technique can be adapted to determine the protein distribution in different tissues other than the brain and model systems other than mice. Begin by washing previously isolated and fixed mouse brain as described in the manuscript.
Then transfer the brain to a 15 milliliter reaction tube with 30%sucrose 0.1 molar PB.To cryo protect the brain incubate it at 4 degrees celsius for 48 hours or until it sinks. After that use a a sharp blade to trim the cryo protected brain depending on the area of interest. Then place the brain in a cryo mold and add Optimal Cutting Temperature Compound to embed it making sure to avoid bubbles and orient the brain properly so as to have a coronal plane of cutting.
Place the cryo mold in the minus 80 degree celsius freezer until it is frozen solid. Mount the frozen tissue on the cryo microtome and wait for at least 15 minutes before section to equilibrate it to the microtome temperature. Start cutting the brain into 25 micrometer thick coronal slices.
Use a glass hook to the OCT of the first slice carefully without touching the brain tissue. The OCT will stick to the glass hook. Use the glass hook to transfer the brain slice into the first well.
Collect three adjacent slices per well in a 24 well plate filled with 0.1 molar PB.Store the slices at four degrees celsius until staining for up to two weeks. To prepare the slices for immuno staining use a plastic pipet to remove the PB solution from one well without drawing in the brain slices. Then use a 1000 micro liter pipet 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 pipet to remove the PB solution from the first well. Use a 1000 microliter pipet 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 add 250 microliters of antibody buffer per well to a reaction tube. Then use a 2 microliter pipet to add the appropriate amount of antibody pipetting it directly into the solution and gently pipet up and down several times to mix.
Then vortex this diluted antibody to unsure proper mixing. Working well by well remove the blocking buffer with a plastic pipet and add 250 microliters of primary antibody solution per well. Incubate the plate on a shaker at four degrees celsius overnight.
The following day remove the antibody solution with a plastic pipet. Wash the slices with 300 microliters of washing buffer one per well three times ten minutes each wash on a shaker at room temperature. During the washing, working in the dark, dilute the fluor for a couple second 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 pipet 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 pipet.
Wash the sections three times with washing buffer two in the same way as with washing buffer one. During this washing dilute DAPI stain in 0.1 molar PB to achieve a one 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 pipet use a 1000 microliter pipet 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 cover slip 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 four 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 or one image into FIJI by clicking file and then open. Then use the free hand selection tool to delineate one hemisphere in the DAPI channel.
Click on edit, then selection, 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 single 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 free hand selection tool. After staining with DAPI which stains nuclei brain sections have a punctate pattern and regions with high cell density are brighter than regions with low cell density.
Staining with Mover antibody revealed heterogeneous distribution with bright hot spot areas and dimmer areas throughout the brain where as Synaptophysin revealed more homogenous distribution. An overlay of images showed the differential distribution of red fluorescence indicating Mover protein compared green fluorescence indicating Synaptophysin. After quantification analysis mean fluorescence intensity values for different channels across the hemispheres and across the areas of interest have been determined for both Mover and Synaptophysin.
Ratios of the mover fluorescence values to Synaptophysin fluorescence values show the heterogeneous distribution of Mover with areas of high and low Mover levels relative to Synaptic vesicles. Relative Mover abundance the ratio in one area of interest compared to that across the hemisphere and translated into a percentage showed how much Mover is present in one area of interest relative to the average. Relative Mover abundance was determined for different layers in the sub fields of the hippocampus.
The three regions of interest are quantified by comparing the ratio in the respective layers to the ratio of the corresponding hemisphere. This procedure can easily be adapted and combined with different imaging techniques such as super resolution microscopy to additionally determine the sub cellular distribution of the protein of interest. This technique can also be applied to different model systems such as genetically modified or drug treated animals.
This can allow for a comparative approach across different experimental conditions or even species.
