Immunohistochemical analyses were performed as previously reported [56 (link),57 (link)]. Briefly, mouse brain tissues were placed in 10% neutral buffered formalin overnight. Tissues were embedded with paraffin, sectioning and placed on slides. Deparaffinized sections were stained with 4G8 antibody (#800701, BioLegend, San Diego, CA, USA) for amyloid-β (Aβ). Staining was visualized by using HRP-specific DAB substrate kit with nickel (Vector Laboratories, Burlingame, CA, USA) and mounted in DPX mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA). Image files were imported to ImageJ (NIH) for quantification. Approximately 4–6 slices per animal were used and Aβ was quantified using burden threshold.
4g8 antibody
Manufactured by BioLegend
Sourced in United States
The 4G8 antibody is a monoclonal antibody that recognizes the amyloid-beta (Aβ) peptide, a key component of the amyloid plaques found in Alzheimer's disease. The 4G8 antibody can be used in various research applications, such as immunohistochemistry, Western blotting, and ELISA, to detect and study the Aβ peptide.
Automatically generated - may contain errors
Lab products found in correlation
Dpx mounting medium, by Electron Microscopy Sciences (2 mentions)
Biotinylated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Formalin free tissue fixative, by Merck Group (1 mentions)
Streptavidin biotin blocking kit, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Dab substrate kit with nickel, by Vector Laboratories (1 mentions)
Donkey anti mouse igg conjugated to texas red, by Merck Group (1 mentions)
6 protocols using 4g8 antibody
1
Quantifying Amyloid-Beta Deposition
2
Amyloid Quantification in Mouse Brains
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Mouse brains were hemi-sectioned and halves placed directly into 10 ml of Formalin-Free Tissue Fixative (Sigma, St. Louis MO) and stored at 4 °C until prepared for sectioning. To assess the extent of brain neuropathology, paraffin-embedded hemibrains were serially sectioned at 10 µm intervals. Twelve matched slices from each brain were taken from equivalent regions (plates 12–16, ref.50 ), deparaffinized and processed for amyloid detection. For immunohistochemistry, sections were washed in Tris-buffered saline (TBS), blocked with a streptavidin/biotin blocking kit (ThermoFisher Scientific, Waltham, MA), and then incubated in TBS containing 0.5% Tween and 5% donkey serum for 30 min. Tissues were incubated in 4G8 antibody (1:1000; Biolegend) diluted in blocking solution at 4 °C overnight. After washing, sections were incubated in biotinylated secondary antibody (Jackson Immunoresearch; dilution 1:200), followed by incubation with Dylight 549 Streptavidin. Fibrillar Aβ deposits were visualized using Thioflavin S (Thio-S), following previously described methods51 (link). Briefly, mouse brain sections were washed with TBS and stained for 10 min with a solution of 0.5% Thio-S in 50% ethanol. Finally, sections were washed in 50% ethanol and TBS, dried, and coverslipped using Vectashield (Vector Laboratories, Burlingame, CA).
3
Immunohistochemical Analysis of Amyloid-Beta
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Immunohistochemical analyses were performed as previously reported.47 , 48 Mouse brain tissues were placed in 10% neutral buffered formalin overnight, followed by paraffin embedding and sectioning. Sagittal sections of the brain were stained for Aβ with the 4G8 antibody (#800701, Biolegend, CA, USA). Sections were visualized by using DAB substrate kit with nickel (Vector Laboratories, CA, USA) and mounted using DPX mounting medium (Electron Microscopy Sciences). Approximately 4–6 slices per animal were taken and analyzed in the ImageJ (NIH) software and quantified using burden threshold.
4
Quantitative Aβ Immunohistochemistry in Mouse Brain
5
AY51 Modulation of SARS-CoV-2 Pathogenesis in APP/PS1 Mice
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At 8 weeks of age, female APP/PS1 double transgenic mice were treated with AY51 (administered intranasally at a dosage of 500 μg/kg body weight, 5 μL) and subsequently infected intranasally with SARS-Cov-2 pseudovirus (108/mL, 2 μL) (6 mice/group). As a negative control, an empty lentiviral vector (LV-empty pseudovirus) was used. Aβ plaques were detected in cortical and hippocampal sections using the 4G8 antibody (1:1000; Biolegend, 800701) via immunohistochemistry (IHC) at 6 and 13 months of age. Furthermore, the expression of hAPP695wt in AAV-hAPP695 mice and their AAV-vector control counterparts was determined via Western Blot, utilizing the 6E10 antibody. In 10-µm paraffin-embedded brain tissue sections from AAV-hAPP695wt mice and AAV-hAPP695wt/AY51 mice—specifically those containing the hippocampus—the locations of the pseudovirus and AY51 were ascertained by IHC staining using the anti-AY51 antibody and anti-luciferase antibody (1:2000; orb1147993, Biorbyt), respectively. In addition, IHC staining with the 6E10 antibody also validated the expression of hAPP695wt.
6
Colocalization of Aβ oligomers with Aβp and p-Tau
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Double immunofluorescence labelling was performed to determine whether Aβ1 - 42 oligomers co-localized with Aβp in the cortex and hippocampus. Sections were co-stained with 4G8 antibody (1:500; Bio legend, San Diego, CA, USA) and anti-Aβ1 - 42 oligomer nanobody (PrioAD13; 1:500) [21 (link)]. Double immunofluorescence labelling was also performed to determine whether Aβ1 - 42 oligomers co-localized with p-Tau in the cortex and hippocampus using anti-Aβ1 - 42 oligomer nanobody (PrioAD13; 1:500) and AT8 anti-p-Tau (Ser202, Thr205) antibody (Thermo-fisher Scientific, MA, USA). Sections were incubated with primary antibodies overnight at 4°C. Sections were washed three times with TBST then incubated with secondary antibodies: goat anti-llama IgG conjugated to FITC (Bethyl Laboratories, Inc, TX, USA) and donkey-anti-mouse IgG conjugated to Texas red (Sigma-Aldrich, MO, USA) at a dilution of 1:500 for 2 h at RT. For IHC and IF studies, duplicate sections were also stained with secondary antibodies with omission of primary antibodies as negative control. After washing with TBST sections were cover slipped with paramount aqueous mounting medium (Dako, Agilent, Santa Clara, CA, USA), sealed and dried overnight. Finally, images were acquired using the LSM800 Confocal microscope with a standard FITC/Texas Red double band-pass filter set.
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