transgenic mice are sacrificed using carbon dioxide gas , followed by decapitation and the brain extracted .
the other will be submerged in 4% paraformaldehyde ( pfa ) for at least 48 hours at 4c .
after fixation in 4% pfa for at least 48 hours , the hemi brain is transferred directly into 30% sucrose solution and placed at 4c . at the point ,
when the hemi brain sank to the bottom , usually requiring about 48 hours , it is ready to be embedded in oct for cryosectioning . before cryosectioning ,
mount a thin layer of oct on the knob and allow to freeze at -20c .
remove the cerebellum from the rest of the hemi brain and place onto the layer of frozen oct .
immediately cover the hemi brain with oct and allow to slowly freeze at -20c . once the oct embedding the brain has turned opaque , it is ready to be sectioned .
after formic acid treatment , the section will temporarily turn opaque and may appear to shrivel . remove each section into a plate and wash with tx - pbs ( 0.3% triton x-100 in pbs ) for 5 min x 3 with gentle shaking .
block endogenous peroxide blocking 0.5% h2o2 in tx - pbs , rt 30 min with gentle shaking .
block section with 5% non - fat skim milk dissolved in tx - pbs at room temperature for 1 hour with gentle shaking .
incubate sections in primary antibody ( biotinylated 4g8 , 1:500 - 1:2000 , signet ) diluted in tx - pbs containing 5% milk at 4c overnight with gentle shaking . wash section with tx - pbs for 10 min x 3 with gentle shaking .
prepare abc solution following manufacturer 's protocol ( elite abc , vector laboratories , ) .
free - floating brain sections are mounted on gelatin coated slides , to help with tissue adhesion .
sections are then air dried followed by clearing with a series of xylene and mounted in entellen .
plaques are visualized under light microscopy at 40x magnification and are quantified by assessing the mean plaque count per slice for each mouse .
mount 4 to 5 hemi brain sections onto a glass slide and allow to completely air dry prior to staining .
incubate slides in filtered ( 0.2 m filter ) thioflavin s solution ( 1% in 80% of ethanol ) for 15 minutes .
( thioflavin s solution needs to be filtered before each use ) . note : protect thioflavin s from light and protect stained slides from light .
mount slides in aqueous mounting media and allow slides to dry in the dark for at least 2 hour , followed by sealing coverslip with clear nail polish .
analyze slides within one week because the staining will fade with time . 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 - a using biotinylated 4g8 and detected via the abc method .
neuritic plaques are clearly labeled with the antibody and are indicated by the white arrows .
we detected neuritic plaque deposition 2 month old app23xps transgenic mice ( figure 2 ) .
thioflavin s bound a-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
again , using both 4g8 anti - a immunohistochemistry staining and thioflavin s staining , we found that hypoxia facilitated the formation of a containing neuritic plaques ( 4 ) .
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 - a antibody 4g8 and were developed using the abc and dab methods .
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 .
transgenic mice are sacrificed using carbon dioxide gas , followed by decapitation and the brain extracted .
the other will be submerged in 4% paraformaldehyde ( pfa ) for at least 48 hours at 4c .
after fixation in 4% pfa for at least 48 hours , the hemi brain is transferred directly into 30% sucrose solution and placed at 4c . at the point ,
when the hemi brain sank to the bottom , usually requiring about 48 hours , it is ready to be embedded in oct for cryosectioning . before cryosectioning ,
mount a thin layer of oct on the knob and allow to freeze at -20c .
remove the cerebellum from the rest of the hemi brain and place onto the layer of frozen oct .
immediately cover the hemi brain with oct and allow to slowly freeze at -20c . once the oct embedding the brain has turned opaque , it is ready to be sectioned .
after formic acid treatment , the section will temporarily turn opaque and may appear to shrivel . remove each section into a plate and wash with tx - pbs ( 0.3% triton x-100 in pbs ) for 5 min x 3 with gentle shaking .
block endogenous peroxide blocking 0.5% h2o2 in tx - pbs , rt 30 min with gentle shaking .
block section with 5% non - fat skim milk dissolved in tx - pbs at room temperature for 1 hour with gentle shaking .
incubate sections in primary antibody ( biotinylated 4g8 , 1:500 - 1:2000 , signet ) diluted in tx - pbs containing 5% milk at 4c overnight with gentle shaking .
prepare abc solution following manufacturer 's protocol ( elite abc , vector laboratories , ) .
free - floating brain sections are mounted on gelatin coated slides , to help with tissue adhesion .
sections are then air dried followed by clearing with a series of xylene and mounted in entellen .
plaques are visualized under light microscopy at 40x magnification and are quantified by assessing the mean plaque count per slice for each mouse .
mount 4 to 5 hemi brain sections onto a glass slide and allow to completely air dry prior to staining .
incubate slides in filtered ( 0.2 m filter ) thioflavin s solution ( 1% in 80% of ethanol ) for 15 minutes .
mount slides in aqueous mounting media and allow slides to dry in the dark for at least 2 hour , followed by sealing coverslip with clear nail polish .
stained sections should be stored in the dark at 4c . the green fluorescence stained plaques could be visualized with fluorescent microscropy .
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 - a 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 a-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 - a immunohistochemistry staining and thioflavin s staining , we found that hypoxia facilitated the formation of a containing neuritic plaques ( 4 ) .
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 - a antibody 4g8 and were developed using the abc and dab methods .
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 .
immunohistochemistry using the biotinylated labeled 4g8 antibody stains neuritic plaques in ad modeled mice with specificity .
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 4c prior to submerging in the 30% sucrose solution .
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 a and recognized the epitope in the core sequence ( vffae ) . since 4g8
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
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 .
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 .
 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  protein ( a)1 , which is derived from sequential proteolytic cleavage of the beta - amyloid precursor protein ( app ) by -secretase and -secretase . 
 the amyloid hypothesis entails that a-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 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 . 
Protocol
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
Discussion
Disclosures