Total proteins from the hippocampus were extracted using the lysis and quantified using the bicinchoninic acid (BCA) (Sigma-Aldrich, USA) method. The separation of total proteins was conducted using the 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), while separated proteins were transferred to the polyvinylidene fluoride (PVDF) membranes (Invitrogen, USA). The membranes were incubated with 5% non-fat milk for 2 h. Then the membranes were treated with the antibodies against p-Tau (pSer214) (#ab170892, 1:500), Tau46 (#ab203179, 1:2000), BDNF (#ab108319, 1:3000), p-CREB (#ab32096, 1:500), CREB (#ab32515, 1:3000), PDE4B (#ab170939, 1:2000) and β-actin (#HC201-01, TransGen Biotech, China, 1:5000) were imported. Subsequently, the secondary antibody (#RGAM002, 1:2000, Proteintech, USA) was introduced. The exposure of bands was conducted with the ECL solution for quantification.
Rgam002
Manufactured by Proteintech
Sourced in United States
RGAM002 is a laboratory equipment product designed for general research purposes. It functions as a component for conducting various scientific experiments and analyses. The core purpose of this equipment is to facilitate data collection and processing within controlled laboratory settings.
Automatically generated - may contain errors
3 protocols using rgam002
1
Hippocampal Protein Profiling for Neurodegeneration
2
Hyperglycemia-induced ROS Accumulation
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ZIPK collaborates with STAT5A in hyperglycemia-induced ROS accumulation mouse IgG secondary antibody (1:500; RGAM002; Proteintech) and Plus 594-goat anti-rabbit IgG secondary antibody (1:500; RGAR004; Proteintech) in PBS for 1 h at room temperature. After three times wash with PBS, the cell nuclei were stained with 4′,6-diamidino-2phenylindole (DAPI). Images were captured using a confocal fluorescence microscope (Leica, Wetzlar, Germany).
3
Immunofluorescence Imaging of GFAP and NeuN
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After antigen retrieval, permeabilization, and blockage, the paraffin slices were incubated with rabbit anti-GFAP (1:300, 16825-1-AP, Proteintech) and mouse anti-Neun (1:300, 66836-1-Ig, Proteintech) overnight at 4°C. Secondary antibodies, including Alexa Fluor 594 IgG goat anti-rabbit and multi-rAb CoraLite Plus 488-goat anti-mouse (1:1000, RGAM002, Proteintech), were added for 1 h at room temperature. After sealing with an anti-fluorescence quencher, scanning was performed using a Zeiss super-resolution laser scanning microscope LSM900 with Airyscan 2. Seven layers of sliced images (1 μm per layer) were scanned in the optimal signal mode of Airyscan using Airyscan 3D processing, and a maximum intensity projection (MIP) was applied. Considering a slice thickness of 8 μm, the projected image captured almost all slice details.
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