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 .