Rats and control mice were euthanized with an overdose of carbon dioxide and transcardially perfused with PBS. Brain tissue was subsequently harvested and post-fixed overnight in 4% PFA in PBS. Brains were cut in serial sections of 40 μm thickness with a vibrating microtome (Leica). For each sample, six series of sections were sequentially collected in free-floating conditions and permeabilized for 30 min with PBST (0.2% Triton X-100 in PBS) and blocked for 2 h with 5% normal donkey serum in PBST. Antigen retrieval was performed by boiling the sections for 1 min in 10 mM sodium citrate (pH 6) in a microwave. After three rinses with 0.1% Tween 20 in PBS, sections were incubated for 20 min with 10 μM X34 (Sigma) in 0.1% NaOH made in 40% Ethanol washed and incubated overnight at 4 °C with primary antibodies against Aβ: 82E1 (IBL, 1/150) or 6E10 (BioLegend, 1/200). The antibody stained sections were washed three times with 0.2% TritonX100-PBS and incubated with Alexa594 Donkey anti-mouse IgG (Thermo Fisher, 1/300) for 2 h at RT. After final washes with 0.2% TritonX100-PBS, sections were counterstained with TO-PRO (Thermo Fisher, 1/1000) and after three final washes were mounted on super frost microscope slides. Sections were visualized on a Nikon A1R Eclipse confocal system.
A1r eclipse confocal system
Manufactured by Nikon
The Nikon A1R Eclipse confocal system is a high-performance laser scanning microscope designed for advanced imaging applications. It features a resonant scanner for rapid image acquisition and a galvanometer scanner for high-resolution imaging. The system is equipped with multiple laser lines and sensitive detectors to capture detailed fluorescence signals from a variety of samples.
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Lab products found in correlation
3 protocols using a1r eclipse confocal system
1
Immunohistochemical Analysis of Amyloid-Beta in Rat Brain
2
Multiplexed RNAscope Analysis of APOE and TREM2
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RNAscope analysis was performed according to the instructions by the manufacturer on the Manual Fluorescent Multiplex kit v2 (ACDbio, 323,100) on PFA fixed tissue sections. Sections were air dried and incubated with Protease IV for tissue digestion and then incubated with the following probes for 2h at 40°C: Hs-APOE-C3 (ACDbio, 433091), Hs-TREM2-C1 (ACDbio, 420491) as part of the hybridization step. After the amplification step, sections were incubated with the TSA Plus fluorophores (TSA Cyanine Plus Evaluation Kit, Perkin Elmer, NEL744E001KT) for 30min at 40°C. Immediately after all steps were performed, sections were blocked and standard IHC was performed. Images were acquired with the Nikon A1R Eclipse confocal system.
3
Amyloid Immunostaining and Quantification in Rodent Brains
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Rats and control mice were euthanized with an overdose of carbon dioxide and transcardially perfused with PBS. Brain tissue was subsequently harvested and post-fixed overnight in 4% PFA in PBS. Brains were cut in serial sections of 40 μm thickness with a vibrating microtome (Leica). For each sample, six series of sections were sequentially collected in free-floating conditions and permeabilized for 30 minutes with PBST (0.2% Triton X-100 in PBS) and blocked for 2 hours with 5% normal donkey serum in PBST. Antigen retrieval was performed by boiling the sections for 1 minute in 10 mM sodium citrate (pH6) in a microwave. After three rinses with 0.1% Tween 20 in PBS, sections were incubated for 20 minutes with 10 µM X34 (Sigma) in 0.1% NaOH made in 40% Ethanol washed and incubated overnight at 4°C with primary antibodies against Aβ: 82E1 (IBL, 1/150) or 6E10 (BioLegend, 1/200). The antibody stained sections were washed three times with 0.2% TritonX100-PBS and incubated with Alexa594 Donkey anti-mouse IgG (Thermo Fisher, 1/300) for 2 hours at RT. After final washes with 0.2% TritonX100-PBS, sections were counterstained with TO-PRO (Thermo Fisher, 1/1000) and after three final washes were mounted on super frost microscope slides. Sections were visualized on a Nikon A1R Eclipse confocal system.
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