Research reported in this publication was supported by the National Institute of Aging of the National Institutes of Health under award numbers R01AG062840 (to RKS) and R01AG072896 (to RKS). The content is solely the authors&#39; responsibility and does not necessarily represent the official views of the National Institutes of Health. Approximately $200k (100%) of federal funds supported this project. We would also like to thank Dr. Joshua Yang for his assistance with manuscript editing.

All animal experiments were conducted in compliance with the University Laboratory Animal Resources under protocols approved by the University of California, Irvine, Institutional Animal Care and Use Committee. The experiments were performed with male B6C3-Tg(APPswe, PSEN1dE9)85Dbo/Mmjax (APP/PS1) mice (13-month-old, n = 35). The mice were obtained from commercial sources (see Table of Materials).
1. Brain tissue preparation
<ol>
	<li>Anesthetize the mice using a lethal dose of a phenytoin/pentobarbital-based anesthetic (150 mg/kg) injected intraperitoneally (see Table of Materials) following approved animal protocols. Perform cardiac perfusion with ice-cold phosphate-buffered saline (1x PBS) for 5 min at a speed of 5 mL/min to clear the brain vasculature14.</li>
	<li>Harvest brain tissue following the previously published report15, separate into the left and right brain hemisphere and place the right hemi-brain of each mouse into a 15 mL conical tube containing 5 mL of freshly prepared 4% paraformaldehyde&#160;(PFA)&#160;solution in 1x PBS for 72 h at 4 &#176;C. 
	NOTE: The brains may be immersion-fixed from 24-72 h depending on the antigen being studied. The left hemi-brain, without the cerebellum, can be snap-frozen in liquid nitrogen and then stored at -80 &#176;C, followed by processing for biochemical assays such as ELISA and biochemical A&#946; detection16. 
	CAUTION: PFA is a probable carcinogen, and skin contact with PFA may lead to allergic skin symptoms. Use nitrile or butyl gloves, masks, and eye protection for handling it, and prepare under a chemical fume hood.</li>
	<li>After incubation in 4% PFA, incubate the hemi-brains sequentially in 5 mL of 10%, 20%, and 30% sucrose solutions prepared in 1x PBS for 24 h, each at 4 &#176;C until the brain tissue sinks to the bottom of the conical tube.</li>
	<li>After incubation in the 30% sucrose solution, remove the hemi-brain, gently dab the brain on a filter paper to remove the excess sucrose solution, and freeze the fixed hemi-brain in powdered dry ice for 30 min. Store the frozen hemi-brain in well-labeled aluminum foils at -80 &#176;C until cryosectioning. 
	NOTE: In the current protocol, the hemi-brains were stored at -80 &#176;C for 6-8 months.</li>
	<li>Section the frozen hemi-brain into 20 &#181;m thick sections using a cryostat (see Table of Materials). 
	&#8203;NOTE: For the current protocol, the hemi-brains were sectioned into sagittal sections, and if needed, coronal sections can also be prepared17. Step 2 is for A&#946; immunofluorescent staining on fixed and cryoprotected brain tissue samples.</li>
</ol>
2. Immunofluorescence
<ol>
	<li>Place the sagittal brain tissue sections (step 1.5) into a 24-well plate (up to six mouse brain sections per 300 &#181;L per well). Wash for 5 min with 1x PBS three times at room temperature (23 &#176;C) by placing the plate on a shaker with gentle swirling.</li>
	<li>Incubate the brain tissue sections with 70% formic acid in dH20 at room temperature for 10 min. 
	CAUTION: Formic acid is corrosive, so avoid skin and eye contact.</li>
	<li>Wash the brain tissue sections for 5 min with dH20 three times at room temperature.</li>
	<li>Block non-specific binding with 0.5% bovine serum albumin (BSA) and 0.3% TritonX 100 (see Table of Materials) in 1x PBS at room temperature for 1 h.</li>
	<li>Incubate the brain tissue sections with a fluorophore-labeled primary antibody (6E10, see Table of Materials) diluted (1:1000) in 1x PBS containing 0.3% TritonX 100 at 4 &#176;C for 24 h. 
	NOTE: Since the primary antibody is fluorophore-conjugated, the plate needs to be covered from this step onward, or all work must be done in a dark room to maintain fluorophore effectiveness.</li>
	<li>Wash the brain tissue sections for 10 min with 1x PBS three times at room temperature.</li>
	<li>Mount the brain tissue sections onto positively-charged glass slides (see Table of Materials) that are well labeled (label information on the slide is based on preference), after a brief wash with dH20 to remove remaining salts. Let the slides air dry in the dark. 
	NOTE: The brain sections must be mounted carefully to avoid folds and rips, impacting data quantification. In case of rips and/or folds that lie in the region of interest and can interfere with data quantification, re-staining and re-mounting are recommended.</li>
	<li>Mount the brain tissue sections with an aqueous mounting media (see Table of Materials) and place the glass coverslip over the tissue. Seal the ends of the coverslip with clear nail polish and store the slides in a slide box at 4 &#176;C until imaging. 
	&#8203;NOTE: In the current protocol, the slides were imaged within 1 month after staining.</li>
</ol>
3. Imaging
<ol>
	<li>Image the 6E10-stained brain sections using a fluorescence (epifluorescence or confocal) microscope (see Table of Materials), that has a 2x objective to capture the entire brain tissue section in one image and is equipped with the appropriate filter (GFP in this work). 
	NOTE: Imaging settings must be consistent across different slides.</li>
	<li>Save the captured images as a TIFF file or as required and open them in the image analysis software (see Table of Materials) as described in step 4.2 below for 6E10 quantification. 
	&#8203;NOTE: Include a scale bar before capturing the image to quantify in the image analysis software, ImageJ.</li>
</ol>
4. Full-region of interest analysis
NOTE: The present work&#39;s two regions of interest are the hippocampus and the iso-cortex. Full-region of interest analysis represents the analysis of the entire iso-cortex (referred to as the cortex going forward) or the hippocampus in the imaged brain tissue section.
<ol>
	<li>Download the image analysis software (see Table of Materials) and start the software once installed.</li>
	<li>Once the software runs, click on File | Open | Choose the image to be analyzed.</li>
	<li>Click on Analyze | Set scale| Click to remove scale.&#160;Select the&#160;Straight&#160;tool from the software tool bar and draw a straight line along the length of the scale bar. Click on&#160;Analyze | Measure. Note the length or distance of the scale bar in pixels. Click on&#160;Analyze | Set scale.
	<ol>
		<li>In the pop-up window, enter the distance in pixels, the known distance of the scale bar (in &#181;m in this case), and the unit of length as &#181;m. Check&#160;Global&#160;to apply the new scale setting to all following images if multiple images are processed. Click&#160;OK&#160;to apply the settings. 
		NOTE: It is always recommended to check if the accurate scale is applied to the image before further analysis.</li>
	</ol>
	</li>
	<li>To set the desired measurement to the area of the section, go to Analyze | Set measurements | Select the Area and Display Label boxes. Check that the image being analyzed is selected under Redirect to.</li>
	<li>For ease of hippocampus or cortex visualization, go to Image | Adjust | Brightness/Contrast. Drag the Maximum slide bar gradually to the left to increase tissue clarity until the brain regions of interest are identifiable. 
	NOTE: Do not apply this setting to avoid false measurements during analysis, instead proceed to the next step.</li>
	<li>Use the Polygon selection or Freehand selection tool to outline the hippocampus region. Click on the Reset option of the Brightness/Contrast settings once the hippocampus is outlined to revert to the original brightness. 
	NOTE: The steps must be repeated separately for the cortical region.</li>
	<li>To measure the total tissue area of the selected region, click on Edit | Clear outside. Once the selected region is the only image on-screen, click on Analyze | Measure to obtain the total tissue area analyzed in a pop-up window. Save the data in an Excel file for later use.</li>
	<li>To measure the 6E10-positive area, go to Image | Adjust | Color threshold. A pre-made filter under the Thresholding method usually provides desired results highlighting the strongest signals in red. 
	NOTE: The optimal threshold selection will depend on the background of the image and the staining intensity. Select a threshold setting that picks up the stain and not the background.</li>
	<li>After selecting the appropriate threshold, check Dark background. This will highlight the A&#946; spots (the stain of interest) on a black background. Click on Select | Original | Select, giving the dark signals (the A&#946; deposits) on a white background. Click on Analyze | Analyze particles and click OK when the pop-up window generates.</li>
	<li>Copy the summary output generated by the software by clicking on Edit | Copy. Paste into the previously started Excel file with respective labels. This is the 6E10-positive area in the selected regions of interest (either hippocampus or cortex). 
	&#8203;NOTE: In Excel, there will be a column for the total 6E10-positive area (step 4.10) and the total tissue area (step 4.7).</li>
	<li>Calculate the 6E10-positive area (%) as follows16: (Total 6E10-positive area/Total tissue area analyzed) x 100.</li>
</ol>
5. Sub-region of interest analysis
NOTE: Sub-region of interest analysis represents the analysis of a part of the cortex or the hippocampus in the imaged brain tissue section.
<ol>
	<li>Download the image analysis software and start the software once installed.</li>
	<li>Once the software runs, click on File | Open | Choose the image to be analyzed.</li>
	<li>Click on&#160;Analyze | Set scale| Click to remove scale.&#160;Select the&#160;Straight&#160;tool from the software toolbar and draw a straight line along the length of the scale bar. Click on&#160;Analyze | Measure. Note the length or distance of the scale bar in pixels. Click on&#160;Analyze | Set scale.
	<ol>
		<li>In the pop-up window, enter the distance in pixels, the known distance of the scale bar (in &#181;m in this case), and enter the unit of length as &#181;m. Check&#160;Global&#160;to apply the new scale setting to all following images if multiple images are processed. Click&#160;OK&#160;to apply the settings. 
		NOTE: It is always recommended to check if the accurate scale is applied to the image before further analysis.</li>
	</ol>
	</li>
	<li>To set the desired measurement to the area of the section, go to Analyze | Set measurements | select the Area and Display Label boxes. Check that the image being analyzed is selected under Redirect to.</li>
	<li>Adjust the brightness and contrast if the image is too dim and the brain regions (e.g., the hippocampus or the cortex in this case) cannot be easily identified. Use the software toolbar and click on Image | Adjust | Brightness/Contrast and drag the Maximum sliders to the left as needed to increase the tissue visibility. 
	NOTE: Do not apply this setting to avoid false measurements during analysis, instead proceed to the next step.</li>
	<li>Using the Rectangle tool, select the region of interest in the cortex or the hippocampus. Use the toolbar and click on Edit | Selection | Specify, changing the height and width to a pre-defined value. Adjust the box, so it is completely covered by tissue. Reset the brightness/contrast to revert to the original brightness. 
	NOTE: The size of the box used to select the regions of interest must be consistent for all the images. For the present analysis, the box size was either 300 pixels x 300 pixels (equivalent to 1177 &#181;m x 1177 &#181;m) or 400 pixels x 200 pixels (equivalent to 1569 &#181;m x 784 &#181;m).</li>
	<li>Duplicate the selected region of interest by right-clicking the box and clicking on Duplicate. A new window with the selected region will open. Rename the duplicated image to display the region it is located in (e.g., cortex or hippocampus).</li>
	<li>Adjust the duplicated image type by using the toolbar and clicking on Image | Type | 8-Bit to convert the duplicated RGB image to 8-bit to best analyze the plaques. Invert the image by clicking on Edit | Invert.</li>
	<li>To measure the 6E10-positive area, go to Image | Adjust | Threshold. A pre-defined filter under the Thresholding method usually provides the desired results by highlighting the strongest signals in red. 
	NOTE: The optimal threshold selection will depend on the background of the image and the staining intensity. Select a threshold setting that picks up the stain and not the background.</li>
	<li>After selecting the appropriate threshold, select Apply.</li>
	<li>To analyze the 6E10-positive area, use the toolbar and click on Analyze | Analyze particles, ensuring the &#34;Summarize Results&#34; is checked.</li>
	<li>Copy the Summary output with the %Area generated by the software by clicking on Edit | Copy. Paste into the previously started Excel file with respective labels.</li>
	<li>Repeat steps 5.3-5.12 for the different regions in the tissue. Ensure the placement of each box to outline the region of interest is consistent between each image. 
	NOTE: The rotation tool can be utilized if the Rectangle toolbox dimensions cannot fit into the specified region due to tissue curvature.</li>
	<li>To rotate the rectangle, use the toolbar and click on Edit | Selection | Rotate and adjust the rotation degree as needed. Duplicate the image as mentioned in step 5.7 and clear the outside by using the toolbar and clicking on Edit | Clear outside, which clears the 6E10 stains outside the specified rectangle. Proceed with step 5.8 described above.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-beta+and+tau:+the+trigger+and+bullet+in+Alzheimer+disease+pathogenesis.">Amyloid-beta and tau: the trigger and bullet in Alzheimer disease pathogenesis.</a> JAMA Neurology. 71 (4), 505-508 (2014).</li><li>Gremer, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibril+structure+of+amyloid-beta(1-42)+by+cryo-electron+microscopy.">Fibril structure of amyloid-beta(1-42) by cryo-electron microscopy.</a> Science. 358 (6359), 116-119 (2017).</li><li>Ferrero, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First-in-human,+double-blind,+placebo-controlled,+single-dose+escalation+study+of+aducanumab+(BIIB037)+in+mild-to-moderate+Alzheimer's+disease.">First-in-human, double-blind, placebo-controlled, single-dose escalation study of aducanumab (BIIB037) in mild-to-moderate Alzheimer's disease.</a> Alzheimer's &amp; Dementia Translational Research &amp; Clinical Interventions. 2 (3), 169-176 (2016).</li><li>Poon, C. 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The authors have nothing to disclose.

The protocol described herein outlines the procedure for hemi-brain preparation for sagittal sectioning, immunofluorescent staining of A&#946; deposits using the 6E10 antibody on free-floating sections, imaging of the A&#946;-stained brain sections followed by quantification of the A&#946; deposits in the cortex and the hippocampus of mouse brain tissue using an image analysis software. While there are published protocols to quantify A&#946; load in brain tissue sections8,10, this protocol describes the steps involved in the quantification of A&#946; load in the entire iso-cortex (referred to as cortex) and hippocampus in comparison with the A&#946; load in a sub-region of interest in the cortex and the hippocampus, when this may be desired. The correlation between the full- versus sub-regions of interest analyses is also provided.
There are several critical steps in the protocol. First, the protocol described is for 20 &#181;m thick brain tissue sections subjected to free-floating immunostaining, which results in optimal antibody penetration within the tissue section18. The free-floating technique may require the tissue sections to be manually transferred between the different solutions during the immunofluorescent staining, and careful handling of the tissue sections throughout the procedure. This is especially crucial when the tissue sections are immersed in the 70% formic acid solution for antigen retrieval in the current protocol, increasing tissue fragility for thin sections. Alternate approaches to the described protocol include using thicker tissue sections (e.g., 30-40 &#181;m) or using tissue sections that are directly mounted onto positively-charged slides before the immunofluorescent staining. Second, the protocol described herein uses a fluorescently-labeled 6E10 antibody. Besides using the fluorescently-labeled 6E10 antibody, non-fluorescent 6E10 antibodies (e.g., horseradish peroxidase-conjugated 6E10 antibody) can also be used to detect A&#946; load in brain tissue sections, and the current protocol can be adapted to quantify A&#946;-positive immunochemical stains in the brain tissue sections as described previously8. Third, the accuracy of the results for A&#946; load quantification will depend on the appropriate threshold selection in the analysis software, which is dependent on the tissue background and signal intensity. Threshold selection must be performed by the end-user such that only A&#946;-positive stains are selected for quantification. End-user intervention is required to optimize the specific threshold that can be applied to all the images to assure the accuracy of the threshold setting. Fourth, since the sub-region of interest analysis requires selecting a small region of interest in the tissue section, two independent readers were used for this analysis. To maintain independence and blinding during data collection, all the images were number coded; the image analysis sequence was randomized between the readers such that the different readers analyzed different images at any given time, and the data was submitted at the end of each week. Due to the increased likelihood of inter-reader variability in the region of interest selection in the sub-region of interest analysis, the readers were trained using several sample images to optimize region selection in the cortex and the hippocampus before beginning the data collection. This training is crucial to reduce inter-reader variability, and as can be seen (Figure 5A,B), the 6E10-positive area reported by both the readers shows a strong relative agreement in the current study.
The current protocol and the results provide valuable information about the impact of the region of interest size on A&#946;-positive area quantification. A larger region of interest is expected to represent the tissue more than a smaller region of interest. Therefore, sampling a larger tissue is desirable to accurately quantify the A&#946; load in tissues. However, in the case of homogenous A&#946; load distribution within the tissue, a smaller sampling region is generally considered a good representation of the larger tissue under analysis. The current study results confirm this, and the A&#946; load in the entire cortex and the hippocampus was a strong correlate of A&#946; load in a selected sub-region of the cortex and the hippocampus (Figure 5C,D). To further confirm the agreement between the full- and sub-regions of interest analyses, the mean 6E10-positive area in the cortex and the hippocampus were compared, and no difference between the two methods (Figure 5E) was found. This confirms that either of these methods (full- or sub-region analysis) yield comparable A&#946; load measurements.
The current protocol has some limitations. The two methods (full-region versus sub-region analysis) may not always be used interchangeably. The choice of using the full- or sub-region analysis will depend on the regional distribution of A&#946; within the tissue, which is impacted by the age, sex, and strain of the AD mouse model. At 13 months, the A&#946; load is distributed throughout the cortex and hippocampus of the APP/PS1 mice. However, at 6 months, A&#946; deposits are limited to the cortex, and minimal deposits are observed in the hippocampus12. Under such conditions, the full-region of interest analysis may be the desired approach to increase the tissue sampling area and thereby the A&#946; signal. On the other hand, sub-region analysis may be the method of choice when A&#946; load in a specific brain region is of interest, (e.g., the somatosensory cortex). Additionally, at 13 months, the APP/PS1 male mice demonstrate intense 6E10-positive stains, and the immunofluorescent staining results in an excellent signal with a very low background, making the current protocol very suitable for quantification under the given conditions. It is unclear if this quantification method can be applied successfully to less intense staining, and future work will be required to answer this question. The immunofluorescent and image quantification method presented herein detects all forms of A&#946;, including the precursor form13. As a result, if there is an interest in the detection of a specific A&#946; specie (e.g., A&#946;1-40 or A&#946;1-42), antibodies specific to these A&#946; isoforms can be used. Therefore, though the 6E10 immunofluorescent staining and detection method correlated with the measurements of A&#946;1-42 measurements in whole-brain homogenates using ELISA (Figure 5F,G), the correlation was only modest. This can be attributed to the measurement of only A&#946;1-42 using the ELISA and detection of all A&#946; species using the 6E10 immunostaining. The current study uses three readers to assess the agreement and correlation between the full- and sub-regional analyses. Having additional readers may improve the robustness of the study and can further validate the agreements between the two methods presented here. Further, we use the Pearson correlation as a measure of agreement, which is widely used among other methods to describe the agreement between continuous variables19. However, one limitation of using the Pearson correlation to determine agreement is that the two methods used herein may provide related results, but one method may result in overall higher values than the other due to systematic bias. Therefore, Pearson correlation is a good measure of relative agreement19. To increase the robustness of the protocol, additional methods to confirm the absolute agreement, such as comparing the mean 6E10-positive area by the two methods (Figure 5E), can be used19. Taken together, the current protocol compares the A&#946; load detected by immunofluorescent staining and analyzes the full- and sub-regions of interest in brain tissue sections. The results show a strong correlation between these two methods for brain tissue sections derived from 13-month-old APP/PS1 male mice that show abundant A&#946; deposits.

Alzheimer&#39;s disease (AD), the sixth leading cause of death in the United States, continues to be a public health threat, with an estimated 6.2 million Americans living with AD. This is expected to reach 13.8 million by 20601. To date, symptomatic management through medications such as cholinesterase inhibitors and memantine is the primary course of treatment2. AD is characterized by neuropathologic manifestations such as extracellular deposition of amyloid-beta (A&#946;) plaques and intracellular hyperphosphorylated tau accumulation in the form of neurofibrillary tangles3,4. Formed by an endoproteolytic cleavage of the amyloid precursor protein (APP) via beta- and gamma-secretase, A&#946; aggregates to form oligomers and fibrils, leading to neurotoxic effects5. A&#946; has been hypothesized to serve a primary pathological role since the 1980s, and is the therapeutic target of the only FDA-approved disease-modifying therapy for AD6. As a result, transgenic AD mouse models harboring mutations in genes resulting in robust cerebral A&#946; accumulation have been widely used for preclinical AD research since the early 1990s7.
Detection of A&#946; species in these AD transgenic mouse brains is generally done using two immunoassays: enzyme-linked immunosorbent assay (ELISA) and immunostaining. The former assay enables quantitative determination of different A&#946; species and is less time-consuming compared to immunostaining, which requires several sequential tissue processing and imaging steps, including tissue sectioning, immunostaining, imaging, and quantification8. Further, the results obtained following immunostaining are semi-quantitative8. However, the ability to spatially localize A&#946; makes immunostaining an attractive approach for A&#946; detection in brain tissues8.
While using A&#946; immunostaining, several different quantification paradigms have been employed by different research groups. For example, some research groups quantify the A&#946; load in the entire region of interest (cortex or hippocampus), while others quantify the A&#946; load in a specified sub-region of interest (a part of the cortex or the hippocampus)9,10,11. Though the methods for A&#946; detection and quantification have been well established and documented, the impact of the size of the region of interest on A&#946; load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the A&#946; load measurements across the full- and sub-regions of interest using an image analysis software, ImageJ.
The current study used 13-month-old APP/PS1 double transgenic male mice, which express a chimeric mouse/human APP and a mutant presenilin 1, to model early-onset of AD12. A&#946; deposits start developing by 6-7 months of age, and abundant A&#946; accumulation is observed both in the cortex and the hippocampus of these mice by 9-10 months of age12. The transgenic amyloid peptides and holoprotein can be detected by 6E10-immunostaining13, making it a desirable animal model for the present protocol. The procedure covered herein includes brain tissue preparation, immunostaining of free-floating sections, imaging, and quantification of A&#946; load in full- versus sub-regions of interest. The analysis shows a strong correlation between the full- and sub-regional quantification, indicating robust agreement between these two methods in the brain tissue sections derived from 13-month-old APP/PS1 male mice that show abundant A&#946; deposits.

<table><tbody><tr><td>15 mL conical tubes</td><td>ThermoFisher Scientific, MA, USA</td><td>339650</td><td></td></tr><tr><td>24-well plates</td><td>Fisher Scientific, NH, USA</td><td>FB012929</td><td></td></tr><tr><td>Amyloid beta 42 human ELISA kit</td><td>ThermoFisher Scientific, MA, USA</td><td>KHB3441</td><td></td></tr><tr><td>Aqueous mounting media</td><td>Vector laboratories, CA, USA</td><td>H-5501-60</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigmaaldrich, MO, USA</td><td>A2153-50G</td><td></td></tr><tr><td>BZ-X710 Keyence all-in-one fluorescence microscope</td><td>Keyence, IL, USA</td><td>BZ-X710</td><td></td></tr><tr><td>Clear nail poilsh</td><td>User preference</td><td>NA</td><td></td></tr><tr><td>Cryostat</td><td>Leica Biosystems, IL, USA</td><td>Leica CM1860 Cryostat</td><td></td></tr><tr><td>Formic acid</td><td>Sigmaaldrich, MO, USA</td><td>F0507-500ML</td><td></td></tr><tr><td>Glass coverslips</td><td>VWR, PA, USA</td><td>48393-081</td><td></td></tr><tr><td>GraphPad Prism</td><td>GraphPad Software, CA, USA</td><td>Version 8</td><td></td></tr><tr><td>ImageJ 1.51k</td><td>National Institutes of Health, MD, USA</td><td>Version 1.53e</td><td></td></tr><tr><td>Mice</td><td>Jackson Laboratories, ME, USA</td><td>034829-JAX</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigmaaldrich, MO, USA</td><td>P6148-500G</td><td></td></tr><tr><td>Phenytoin/pentobarbital based anesthetic (Euthasol)</td><td>Patterson Veterinary, MA, USA</td><td>07-805-9296</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>Fisher Scientific,&amp;nbsp; NH, USA</td><td>BP661-50</td><td></td></tr><tr><td>Plus (+) microscope slides</td><td>Ted Pella, Inc., CA, USA</td><td>260100</td><td></td></tr><tr><td>Primary antibody (6E10)</td><td>Biolegend, CA, USA</td><td>803013</td><td></td></tr><tr><td>Sucrose</td><td>Sigmaaldrich, MO, USA</td><td>47289</td><td></td></tr><tr><td>Triton X 100</td><td>Sigmaaldrich, MO, USA</td><td>T8787-100ML</td><td></td></tr></tbody></table>

full- versus sub-regional quantification of amyloid-beta load on mouse brain sections

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral A&#946; plaques are widely used to model human AD in mice. Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the A&#946; load in brain tissues derived from AD transgenic mice. Though the methods for A&#946; detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on A&#946; load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the A&#946; load measurements across the full- and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of A&#946; load in full- versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on A&#946;-positive area quantification, and show a strong correlation between the A&#946;-positive area obtained using the full- and sub-regions of interest analyses for brain sections derived from 13-month-old male APP/PS1 mice that show widespread A&#946; deposition.

Here, two different methods are compared to quantify the 6E10-positive area in the hippocampus and the cortex of mouse brain tissues. The two methods are the full-region and sub-region of interest analyses (Figure 1). The full-region of interest analysis, as the name suggests, involves outlining the entire region of interest (in this case, either the iso-cortex or the hippocampus) to determine the 6E10-positive area (Figure 1A,B). The sub-region of interest analysis involves selecting a pre-defined region within the region of interest to determine the 6E10-positive area (Figure 1C,D). The stepwise ImageJ protocol for the two methods is shown in Figure 2, Figure 3, and Figure 4.
This study used three readers; two independent readers performed the sub-region of interest analysis, and the third reader performed the full-region of interest analysis. As seen in Figure 5A,B, there was a strong significant positive correlation (p &#60; 0.0001) between the 6E10-positive area reported by the two readers performing the sub-region of interest analysis (Pearson correlation coefficient r = 0.97 for the cortex and r = 0.96 for the hippocampus). The 6E10-positive areas reported by the two readers for the sub-region of interest analysis were averaged, and the averaged sub-region of interest 6E10-positive area shared a strong significant positive correlation (p &#60; 0.0001) with the 6E10-positive area obtained using the full-region of interest analysis for both the cortex (Pearson correlation coefficient r = 0.96; Figure 5C) and the hippocampus (Pearson correlation coefficient r = 0.95; Figure 5D). The mean cortical- and hippocampal-6E10-positive area obtained by the full-region and sub-region of interest analyses were comparable with no significant difference, confirming the agreement between the two methods (Figure 5E). Further, the insoluble A&#946;1-42 was measured in whole-brain homogenates in a subset of mice and the cortical (Figure 5F) and hippocampal (Figure 5G) 6E10-positive area determined by the full-region of interest analysis was significantly (p &#60; 0.01) correlated with insoluble A&#946;1-42 load using ELISA (see Table of Materials).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63669/63669fig01.jpg" /> 
Figure 1: Full- versus sub-regions of interest selection. Representative images showing the full iso-cortex (cortex) and hippocampus outlined for the full-region of interest analysis in (A) and (B), respectively. Representative images show the selection of sub-region/s of the cortex and hippocampus for the sub-region of interest analysis in (C) and (D), respectively. Scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63669/63669fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63669/63669fig02.jpg" /> 
Figure 2: Protocol for the full-region of interest 6E10-positive area quantification. Image analysis steps showing the original image (A), the image after brightness/contrast adjustment (B), selection of the area of interest (C), clearing (D), threshold adjustment (E), and the final image ready for analysis (F). The numbers in the figure designate the step numbers in the protocol. Scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63669/63669fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63669/63669fig03.jpg" /> 
Figure 3: Protocol for the sub-region of interest 6E10-positive area quantification. Image analysis steps showing the original image (A), the image after brightness/contrast adjustment (B), selection of the area of interest (C), duplication of the image of the region of interest (D), changing the image to 8-Bit and inverting the image (E), threshold adjustment (F), and final image ready for analysis (G). The numbers in the figure designate the step numbers in the protocol. Scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63669/63669fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63669/63669fig04.jpg" /> 
Figure 4: Protocol for the region of interest rotation. Image analysis steps showing the selection of the area of interest and rotation of the selection box to fit the tissue curvature (A), the image of the region of interest after duplication (B), and the image after clearing the outside (non-region of interest) area (C). The numbers in the figure designate the step number in the protocol. Scale bar = 200 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63669/63669fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63669/63669fig05.jpg" /> 
Figure 5: Correlation between the full- and sub-regions of interest analyses. Scatter plots show the correlation between the 6E10-positive area by the two independent readers performing the sub-region of interest analysis for the cortex (A) and the hippocampus (B). A strong positive correlation is observed between the 6E10-positive area resulting from the sub-region of interest analysis and the full-region of interest analysis for both the cortex (C) and the hippocampus (D). There is no statistically significant difference in the mean 6E10-positive area by the full-region of interest and the sub-region of interest analyses in the cortex and the hippocampus (E). A significant correlation is observed between whole-brain homogenate insoluble A&#946;1-42 measurements using ELISA and the full-region of interest analysis for both the cortex (F) and the hippocampus (G). Data were analyzed using the Pearson correlation coefficient, r, in (A-D) and (F-G), and using two-way repeated-measures ANOVA in (E) using a graphing and statistics software. Data are presented as mean &#177; standard error of the mean (SEM) of n = 35 mice in (E), and a two-tailed p &#60; 0.05 was considered statistically significant. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63669/63669fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections at JoVE.com

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

The present protocol describes and compares the procedure to perform a full-region or sub-region of interest analysis of sagittal mouse brain sections to quantify amyloid-beta load in the APP/PS1 transgenic mouse model of Alzheimer's disease.

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of ...

full-versus-sub-regional-quantification-amyloid-beta-load-on-mouse

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.6K Views.  Keck Graduate Institute. Dit protocol vergelijkt de belasting van amyloïde-bèta-plaque in volledige secties van belang en subregio's van belang in hersensecties van APP / PS1 transgene muizen. De methode en resultaten die in dit specifieke protocol worden gepresenteerd, stellen de lezers of onderzoekers in staat om te kiezen tussen de volledige interesseregio of subregio van belanganalyse om een bètabelasting in muizenhersensecties te bepalen. Download om te beginnen de beeldanalysesoftware.Start na de inst...

Video: Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

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 &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-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 A&gamma; 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.

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure change. With the increase of AD patients worldwide, the pathology and treatment of the disease has become a focus in the International Pharmaceutical Industry. Thus, the establishment of the animal model to mimic AD in the laboratory is of great importance.
Here, we describe a detailed protocol for establishing a mimic of AD in a rat animal model though intracerebroventricular injection of amyloid beta protein 25-35 (A&#946;25-35) combined with aluminum trichloride (AlCl3) and anterodorsal thalamic nucleus injection of recombinant human transforming growth factor-&#946;1 (RHTGF-&#946;1) to rats. The related markers of AD were measured, including: spatial memory, neuronal structure and substructure, neuronal A&#946;, and neurofibrillary tangles (NFT) production. This rat model demonstrates spatial memory impairment, neuronal structure and substructure pathological changes, neuron intracellular A&#946; burden, and NFT aggregation, and provides a close mimic of the neuronal structure and function disorder to that of clinical AD patients. Thus, the presented AD rat modelprovides a valuable in vivo tool for exploring neuronal function, neuronal pathology, and drug screening of AD.

This is a protocol to mimic Alzheimer&#39;s Disease in rats by evaluation of spatial memory impairment, neuronal pathological changes, neuronal amyloid beta protein (A&#946;) burden, and neurofibrillary tangles aggregation, induced by the injection of A&#946;25-35 combined with aluminum trichloride and recombinant human transforming growth factor-&#946;1.

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure ...

Behavior

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Medicine

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

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