Marking stem cells and their progeny with an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation of adult stem cells in the brain in vivo. Lineage tracing can reveal novel information about lineage commitment, response to interventions, and multi potency. Advantages of transgenic lineage tracing approaches include specificity of tracing a particular cell lineage, indelible expression of the fluorescent protein regardless of cell turnover or differentiation, low toxicity of doxycyline, conditional activation, and ease of use with common downstream assays on lineage trace tissues including immunostaining and immunofluorescence.
Due to the broad implication of plasticity and cell in the adult brain, the characterization and analysis of adult stem cells will help inform of the study of neurodegenerative diseases, metabolic impacts on brain plasticity, aging, and other related conditions. To begin, warm slides to room temperature and post-fix with ice cold acetone for 15 minutes. Wash slides for five minutes in 1X rinse buffer, shaking at 60 rotations per minute at room temperature between each step.
Permeabilize for standing of nuclear antigens with 0.3%Triton X-100 for 10 minutes at room temperature. Permeabilize for standing of cytoplasmic antigens with 0.3%Tween-20 for 10 minutes at room temperature. Perform antigen retrieval by microwaving slides in 50 to 100 milliliters of 1x DAKO Antigen Retrieval Solution on low for 10 minutes, twice.
Next, rinse with buffer for five minutes at room temperature. incubate slides in 0.3%Typogen Black in 70%ethanol for 20 minutes at room temperature and then wash with rinse buffer. Draw hydrophobic barrier around tissue.
Block for 20 minutes at 37 degrees Celsius with blocking reagent. And incubate with primary antibody diluted in antibody diluent overnight at four degree Celsius. Wash slides for 10 minutes at room temperature.
The next day, incubate sections in Alexa Fluor secondary antibodies for 10 minutes at room temperature. Then wash slides twice in rinse buffer for 10 minutes. Cover brain sections in 100 microliters of one to 500 anti-GFP antibody conjugated to AlexaFluor 488, and incubate overnight at four degrees Celsius to boost the endogenous Fluor4, marking the traced cells.
The next day, wash with rinse buffer two times. And then wash in running DI water for five minutes. Counterstain with 100 nanograms per milliliter 4'6-diamidino-2-phenylindole for five minutes.
Then wash in running DI water for five minutes. Add a drop of fluorescent safe mounting medium on top of each tissue section and seal with a 22 millimeter by 22 millimeter coverslip. Imaging is recommended on the day of mounting to ensure optimal signal.
To begin fixation and sectioning following transcardial perfusion post-fix brains in 4%PFA overnight at four degrees Celsius. And then again for one hour at room temperature. Wash brains in 1x PBS for an hour twice at room temperature.
Cut the brain into one millimeter thick sections using the sagittal brain block and place into five milliliter micro centrifuge tubes containing 1x PBS. For methanol pre-treatment, start by washing with 1x PBS for one hour twice at room temperature. Wash in 50%methanol for one hour at room temperature.
Wash in 80%methanol for one hour at room temperature. Wash in 100%methanol for one hour, twice, at room temperature. Bleach samples with 5%hydrogen peroxide in 20%dimethyl sulfoxide and methanol at four degrees Celsius overnight.
After bleaching, wash samples in methanol for one hour twice at room temperature. Wash in 20%dimethyl sulfoxide and methanol for one hour twice at room temperature. Wash in 80%methanol for one hour at room temperature.
Wash in 50%methanol for one hour at room temperature. Wash in PBS for one hour twice at room temperature. Wash in PBS 0.2%Triton X-100 for one hour twice at room temperature.
For immunostain of clearing brains, begin by incubating samples in 1x PBS, 0.2%Triton X-100, 20%DMSO, 0.3 molar glycine, at 37 degrees Celsius overnight on an orbital shaker. Block in 1x PBS, 0.2%Triton X-100, 10%DMSO, 6%goat serum at 37 degrees Celsius for three days on an orbital shaker. Wash samples in 1x PBS, 0.2%Tween-20, 10 micrograms per milliliters heparin sodium salt from porcine mucosa for one hour twice at 37 degrees Celsius.
Next, incubate in 1x PBS, 0.2%Tween-20, 10 micrograms per milliliters heparin, 5%DMSO, 3%goat serum containing one to 500 concentration of rabbit anti GFP AlexaFluor 488 at 37 degrees Celsius on an orbital shaker for two days. Wash samples in 1x PBS, 0.2%Tween-20 with 10 micrograms per milliliter heparin for one hour at 37 degrees Celsius on an orbital shaker three times and then once a day for two days. Incubate samples overnight in one milliliter of 50%Tetrahydrofuran and water.
Incubate samples for one hour in one milliliter of 80%THF Tetrahydrofuran and water. Incubate samples twice for one hour in 100%Tetrahydrofuran. Dry samples with a sterile wipe and incubate in dichloromethane until they sink to the bottom of the vial.
Do not incubate for more than 60 minutes. Incubate samples in one milliliter of dibenzyl ether until clear. Store samples in dibenzyl ether at room temperature until ready to image.
To image immunostained brain sections, analyze slides via confocal microscopy, where co-expression of multiple antibodies can be confirmed. We use a Leica Stellaris five microscope with HyD S hybrid detectors. To image cleared brains, use an inverted confocal microscope with an automated stage and automated tiling function.
Start by laying a cleared brain section flat against a glass bottom dish in submerge in dibenzyl ether. A noticeable difference in fluorescent intensity across different brain regions and various cell types was observed following a pulse chase period in the lineage traced brains. Choroid epithelial cells in the choroid plexus had a thick cell membrane and strong green fluorescent signal indicating they derived from Tert positive cells which was easily distinguishable from the surrounding cells.
The other smaller cell types in the choroid plexus stroma had a weaker GFP signal. In the cerebellum, Bergmann glial cells expressed higher levels of GFP than basket cells, and variation in GFP expression also existed between individual basket cells. For factory sensory neuron axons within the glomerular layer of the olfactory bulb also expressed high levels of GFP Olfactory sensory neuron axons that fill the glomerular layer of the olfactory bulb expressed high levels of membrane tomato, or red fluorescence when compared to most other brain regions even when the same neurons are GFP positive indicating that some cells appeared to lose mTomato signal more slowly during lineage tracing.
In contrast, in the choroid plexus GFP positive cells expressed low mTomato. In most other brain regions, tomato signals expressed at low level across most cell types with endothelial cells being some of the only cells whose fluorescent signal was bright enough to stand out distinctly from the red fluorescent background of the brain tissue. The most important thing to remember is that the fluorescent signal in cleared brains fades faster than most other immuno assays.
Remember to image your samples as soon as possible and within seven days. Following, imaging of the brain slices or clear brains, colorization of markers with the GFP signals and our quantification of Fluorence can be undertaken using software programs.
