The overall goal of this procedure is to prepare ad model mice brains for detection of retic plaques using 4G eight immuno staining and theof flavin S staining. This is accomplished by first extracting the mouse brain from the skull and passively perfusing in 4%PFA, followed by submerging in 30%sucrose solution cryo section, the brain's longitudinally to a 30 micron thickness. Next stain eretic plaques using the four GA antibody and visualized by the DAB method.
In addition, stain eretic plaques by theof flavin S staining, and visualize using fluorescent microscopy. Ultimately, results obtained can show retic plaque deposition in brain tissues through an antibody-based detection and staining with theof flavin S die. The main advantage of these techniques over existing methods is that 4G eight staining and theof flavin S are highly selective for retic plaques containing a beta proteins.
Although this method we shown here is to detect a beta plaque in Alzheimer disease transgenic mice, this method can also be detect to detect a beta plaque in Alzheimer disease. Human brain tshirts Sacrifice the transgenic mice using carbon dioxide gas, followed by decapitation, extract the brain and dissect it longitudinally at the center. Flash freeze half of the brain on dry ice for biochemical analysis, submerge the other half in 4%paraform aldehyde for at least 48 hours at four degrees Celsius.
Transfer the Hemi Brainin to 30%sucrose solution at four degrees Celsius. Initially, the Hemi brain will float in the sucrose solution, but after 24 to 48 hours, it sinks to the bottom before cryosectioning mount a thin layer of OCT on the knob and allow it to freeze at minus 20 degrees Celsius. Remove the cerebellum from the rest of the Hemi Brainin.
Place the remainder onto the layer of frozen OCT immediately cover it with OCT and allow it to slowly freeze at minus 20 degrees Celsius. Set the cryostat to section thicknesses of 30 microns and collect each slice into a container with Domo solution. Treat each free floating section with 88%formic acid for 15 minutes.
The sections will appear to shrivel. Then transfer each section to a plate and wash three times with Triton XPBS for five minutes with gentle shaking. Attenuate the endogenous activity with 0.5%hydrogen peroxide in Triton XPBS for 30 minutes at room temperature with gentle shaking.
Then wash the sections three times with Triton XPBS for 10 minutes under gentle shaking block sections with 5%non-fat skim milk dissolved in Triton XPBS at room temperature for one hour with gentle shaking. Continue to incubate sections in primary antibody diluted in Triton XPBS containing 5%milk at four degrees Celsius overnight with gentle shaking. Wash the samples three times with trite next PBS for 10 minutes.
Now prepare a b, C solution according to the manufacturer's protocol and incubate sections in the A B, C mixture for 30 minutes with gentle shaking, followed by three washes with trite next PBS for 10 minutes. Incubate sections in freshly prepared DAB mixture for five to 10 minutes when a brownish color develops on each section, wash three times with trite next PBS for 10 minutes with gentle shaking. Mount the free floating brain sections on gelatin coated slides and air dry clear with a series of xylene treatments and mount in lin.
Visualize the plaques under light microscopy at 40 x magnification and quantify mean plaque Count per slice for each mouse extract brain tissue from mice as shown earlier. Mount four to five hemi brain sections onto a glass slide and air dry completely dehydrate samples in 70%Ethanol for one minute, followed by 80%ethanol for one minute. Next, incubate slides in 0.1%filtered THEOF Flavin S solution for 15 minutes.
Taking care to protect Theof Flavin S solution and stain slides from light wash slides with 80%ethanol for one minute, 70%ethanol for one minute and distilled water twice. Now mount the slides in aqueous mounting media and air dry in the dark for at least two hours, followed by sealing the cover slip with clear nail polish. Store the stain sections in the dark at four degrees Celsius.
Finally, visualize green fluorescent stained plaques with fluorescent microscopy within one week. In our recent study on the efficacy of the anti-epileptic drug and mood stabilizer, valproic acid to inhibit retic plaque formation, we use the above described immuno staining and theof flavin s staining procedures to identify retic plaques in ad model mice. A typical nine month old A PP 23 transgenic mouse is stained with anti A beta using BIOTINYLATED 4G eight and detected via the A BC method.
Neuro retic plaques are clearly labeled with the antibody and are indicated by the white arrows. Here theof flavin S staining detects retic plaque deposition in two month old A PP 23 times PS transgenic mice Once master this technique can be completed in three days if performed properly. While attempting this procedure, it is important to completely fix the brain in 4%PFA, and ensure that the brain completely sinks to the bottom of the 30%sucrose solution.
Following this procedure, other methods like immuno standing for tau can be performed in order to answer additional like tau hyper sation and a neuronal f.
