Sera from mice immunized with AV-1980R/A, as well as injected with AdvaxCpG were screened for the ability to bind to human tau tangles using 40 µm brain sections of formalin-fixed cortical tissues from severe AD cases (generously provided by the UC Irvine Alzheimer’s Disease Research Center (ADRC) Tissue Repository) using immunohistochemistry as described previously62 (link). In addition, AD brain sections were stained with several commercial antibodies: anti-human tau (Agilent, CA), anti-phospho tau [pS199; pS202; pS396; pS404] (all from Abcam, UK) and Amylo-Glo (Biosensis, Australia). Sections were imaged using an Olympus FX1200 confocal microscope.
Fx1200 confocal microscope
Manufactured by Olympus
The FX1200 confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a high-resolution optical system, enabling detailed observation and analysis of biological samples. The FX1200 is capable of capturing clear, high-quality images with excellent contrast and resolution.
Automatically generated - may contain errors
10 protocols using fx1200 confocal microscope
1
Tau Tangle Binding Assay in AD Brain
2
Tau tangle binding assay using AD brain
3
Visualizing Amyloid-β Deposition in 5XFAD Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Then, 40-μm brain sections from 8 month old 5XFAD mice were stained with rabbit anti-AβpE3 [Pyro Glu3] antibody (SYSY, 1:200) and 6E10 antibody (BioLegend, 1:1000), as described in [46 (link)]. Immunofluorescent sections were visualized and captured using an Olympus FX 1200 confocal microscope. Representative images represent confocal Z-stacks (12 slices at 1.79-micron step intervals) taken at 40× magnification, and Z-stacks (12 slices at 1.51-micron step intervals) taken at 20× magnification [46 (link)].
4
Quantifying α-Synuclein Inclusions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescent immunohistochemistry followed standard protocols, as detailed in Supporting Information. Sections were imaged in a blinded manner using an Olympus FX1200 confocal microscope, with identical laser and detection settings across a given immunolabel. Immunoreactive cells and α‐syn inclusions were hand‐counted by a blind observer, and sample coding was revealed once all values were obtained.
5
Visualizing Tau Tangles and Amyloid Plaques
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following perfusion, one hemisphere from each mouse was postfixed in 4% paraformaldehyde for 48 h then stored in PBS + 0.05% sodium azide. Fixed half-brains were placed in 30% sucrose for at least 48 h before being cut in the coronal plane (40-μm sections) using a freezing sliding microtome. Brain sections were rinsed in PBS before blocking in PBS + 0.05% Triton-X with 10% goat serum for 1 h. First, samples were stained with Amylo-Glo™ RTD Amyloid Plaque Stain Reagent (Biosensis, Australia) for 15 min, washed three times, and then incubated in pS199 (Abcam, UK, 1:1000) and PHF-1 (gift from Dr. Peter Davis, 1:1000) phospho-tau primary antibodies at 4 °C overnight. The next day, sections were washed three times with PBS and placed in appropriate Alexa Fluor-conjugated secondary antibody solutions at room temperature for 1 h. Sections were rinsed three additional times, mounted onto slides, and coverslipped using Fluoromount-G. For confocal microscopy, immunofluorescent staining was performed on equivalent brain sections and imaged on the Olympus FX1200 confocal microscope. Tau tangles and β-amyloid plaques were visualized using Z-stack maximum-projection images taken through the entire depth of the section at 1-μm intervals.
6
Visualizing Microglial Dynamics via Confocal Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescent sections were visualized and captured using an Olympus FX 1200 confocal microscope. Representative images of microglia in the fornix after PBS and LPS treatment represent confocal Z-stacks (14 slices at 1.77-micron step intervals) taken at 40× magnification. Half brain stitch images represent confocal Z-stacks (11 images at 2.39-micron step intervals) taken at 10× magnification. Whole brain stitch images represent confocal Z-stacks (11 images at 2.81-micron step intervals) taken at 4× magnification. Representative images of engrafted microglia in the hippocampus and cortex represent confocal Z-stacks (14 slices at 1.77-microns step intervals) taken 40× magnification. Representative images of engrafted microglia in PLX3397 30 d on/30 d off represent confocal Z-stacks (12 slices at 1.51-microns step intervals) taken at 20× magnification. Stitch was performed using Fluoview FV31S-DT software. Three sections were imaged per region per animal for each quantification. In some cases, brightness and contrast settings of confocal images were adjusted to reveal fine structures and morphology in representative images. Importantly, no such changes were made to any images used for quantification.
7
Screening Amyloid and Tau Pathology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sera from mice immunized with AV-1959R/A, AV-1980R/A, and mixture of AV-1959R/A and AV-1980R/A, as well as control mice injected with AdvaxCpG only, were screened for the ability to bind to human Aβ plaques or/and tau tangles using 40-μm brain sections of formalin-fixed cortical tissues from a severe AD case (received from Brain Bank and Tissue Repository, MIND, UC Irvine) using immunohistochemistry, as described previously [20 (link)–22 (link)]. In addition, brain sections were stained with anti-Aβ (beta-amyloid (1–42), 1:250, Invitrogen, CA) and humanized anti-tau (Armanezumab, 1:1000; Institute for Molecular Medicine, CA) antibody as positive controls. Sections were imaged using an Olympus FX1200 confocal microscope, with identical laser and detection settings across a given immunolabel.
8
Confocal Microscopy Imaging Protocol
9
Immunostaining of Brain Tissue Slices
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fixed half brains were first cryoprotected in a 30% sucrose and 0.05% NaN3 solution in 1X PBS for 72 h. Tissue was then sectioned into 40 μm thick slices on a freezing microtome (Leica SM 2010R), and stored in 0.05% NaN3 solution in 1X PBS as free floating wells. For staining, tissue was blocked for 1 h in 1X PBS, 0.2% Triton X-100, and 10% goat serum. Immediately following blocking, sections were placed in primary antibodies diluted in 1X PBS and 1% goat serum and incubated overnight on a shaker at 4 °C. Sections were labeled with combinations of anti-Ku80 (1:250; Abcam ab79220), anti-Iba1 (1:200; Wako 019–19,741), anti-P2RY12 (1:200; Sigma HPA014518) and mounted with DAPI Fluoromount (SouthernBiotech). Immunofluorescent sections were then visualized and captured using an Olympus FX1200 confocal microscope. Images represent confocal Z-stack taken with equivalent laser and detection settings.
10
Fluorescent Immunohistochemistry Quantification
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!