Department of Neuroscience, The University of British Columbia
Ly, P. T., Cai, F., Song, W. Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model. J. Vis. Exp. (53), e2831, doi:10.3791/2831 (2011).
Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid β protein (Aβ)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by β-secretase and γ-secretase. The amyloid hypothesis entails that Aγ-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of Aγ neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.
1. Fixation of mouse brains and cryosectioning
2. Immunohistochemistry procedure for neuritic plaques (free floating sections)
3. Thioflavin S staining procedure for detection of neuritic plaques
4. Representative Results
In our recent study on the efficacy of the anti-epileptic drug and mood stabilizer valproic acid (VPA) to inhibit neuritic plaque formation, we used the above described immunostaining and thioflavin S staining procedures to identify neuritic plaques in AD model mice (3). Figure 1 represents a typical 9 month old APP23 transgenic mouse, stained with anti-Aβ using biotinylated 4G8 and detected via the ABC method. Neuritic plaques are clearly labeled with the antibody and are indicated by the white arrows. Using thioflavin S staining, we detected neuritic plaque deposition 2 month old APP23xPS transgenic mice (Figure 2). Thioflavin S bound Aβ-containing neuritic plaques appears green under fluorescence microscopy (indicated by white arrows). We showed that with VPA treatment, neuritic plaque formation is significantly reduced (3). In another study, we studied the molecular link underlying hypoxia and AD pathogenesis. Again, using both 4G8 anti-Aβ immunohistochemistry staining and thioflavin S staining, we found that hypoxia facilitated the formation of Aβ containing neuritic plaques (4).
Figure 1. Immunohistochemical analysis of neuritic plaque formation in AD transgenic mice. APP23 mice at the age of 9 months were sacrificed and were dissected, fixed, and sectioned. Neuritic plaques in the hippocampal were detected using an anti-Aβ antibody 4G8 and were developed using the ABC and DAB methods. The plaques were visualized by light microscopy with 40X. White arrows point to Aβ neuritic plaques.
Figure 2. Thioflavin S staining of neuritic plaques in AD modeled mice. APP23xPS45 double transgenic mice at 8 weeks old were sacrificed and were dissected, fixed, and sectioned. Neuritic plagues in the hippocampal are were confirmed using thioflavin S fluorescent staining and visualized by microscopy at 40X magnification. White arrows indicate thioflavin S stained neuritic plaques.
Immunohistochemistry using the biotinylated labeled 4G8 antibody stains neuritic plaques in AD modeled mice with specificity. The staining outcome allows quantification and comparison of plaque load between different treatment groups. There are several crucial steps that could affect the outcome. Because the hemi brains are not perfused prior to extraction from the skull, care should be used during the extraction process in order to prevent damages to the hemi brain. Moreover, since the hemi brain is passively perfused, we recommend incubating it in 4% PFA for at least 48 hours at 4°C prior to submerging in the 30% sucrose solution. Alternatively, transcardial perfusion could be done prior to extracting the brain. If the brain is fixed properly, it should have a rubbery texture. In the case where the brain is not fixed properly, the sections will easily tear in the D'Olomos solution or during the staining procedures.
The 4G8 monoclonal antibody is reactive to amino acid residues 17-24 of Aβ and recognized the epitope in the core sequence (VFFAE). Since 4G8 is derived from mouse species, it tends to give a higher background staining. Thus, we chose to incubate the sections at the indicated dilution with considerations to achieving a true signal while minimizing background staining.
In addition to immunostaining for neuritic plaques detection using the 4G8 antibody, a thioflavin S staining method is also used to identify plaques. Thioflavin S is a homogenous dye mixture that results from methylation of dehydrothiotoluidine with sulfonic acid. Thioflavin S non-selectively binds beta sheet contents of proteins, such as those in amyloid oligomers. Upon binding, thioflavin undergoes a characteristic blue shift of its emission spectrum. Conversely thioflavin S binding to the monomeric forms will not elicit a blue shift and could not be detected with florescent microscope. Thioflavin S staining provides a quick alternative to screen for amyloid as the intensity of fluorescence allows good visualization of small amounts of amyloid deposits. However, one drawback about thioflavin S staining is the lack of specificity. Many other tissue components including containing extensive beta sheets, such as fibrinoids, hyaline, keratin, etc, have a rather affinity for this dye.
All experiments were conducted in accordance with The University of British Columbia Animal Care and Use committee and CIHR guidelines.
This work was supported by the Canadian Institutes of Health Research (CIHR), the Townsend Family, and Jack Brown and Family Alzheimer's Research Foundation (to W. S.). W. S. is the holder of the Canada Research Chair in Alzheimer's Disease. P. T.T. L. is supported by the Natural Sciences and Engineering Research Counsel and the Michael Smith Foundation for Health Research Scholarship.
|Tissue-Tek O.C.T. Compound||Sakura Finetek||4583|
|88% formic acid||Fisher Scientific||A118P-500|
|Triton X-100||ICN Biomedicals||807426|
|Biotin Labeled 4G8 Antibody||Cedarlane Labs||SIG-39240-500|
|Elite ABC kit||Vector Laboratories||PK-6100|
|Imm PACT DAB||Vector Laboratories||SK-4105|
|Entellan||Electron Microscopy Sciences||65037-71|
|Superfrost* Plus Microscope Slides||Fisher Scientific||12-550-15|
|MX35 Premier microtome blade||Thermo Fisher Scientific, Inc.||3052835|