The hippocampus sections (30 μm, 6 offspring rats per experimental group, 3 sections per animal) were incubated with 3% H2O2 for 25 minutes at room temperature in a wet box to inactive endogenous hydrogen peroxide enzymes. The sections were incubated with relative primary antibodies—anti-HDAC2 (1:200, ab32117, Abcam, Cambrige, UK), anti-Neun (1:200, ab177487, Abcam, Cambrige, UK), anti- p-CREB (1:100, ab32096, Abcam, Cambrige, UK)(dissolved in 1% goat serum albumin in phosphate buffered saline) at 4°C overnight. Then, the sections were exposed to the green fluorescent-conjugated secondary antibody and the red fluorescent-conjugated secondary antibody (1:500, TransGen Biotech, Beijing, China). Finally, the sections were wet mounted and immediately viewed using a fluorescence microscope (400X).
Green fluorescent conjugated secondary antibody
Manufactured by Transgene
Sourced in China
The green fluorescent-conjugated secondary antibody is a laboratory reagent designed to facilitate the detection and visualization of target proteins or biomolecules in various experimental applications. It functions by binding to primary antibodies that have been used to label the target of interest, allowing for the amplification of the fluorescent signal and improving the sensitivity of the detection process.
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2 protocols using green fluorescent conjugated secondary antibody
1
Hippocampal Protein Expression Quantification
2
Hippocampal Protein Expression Analysis
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Paraffin-embedded hippocampi (n = 6) were cut into 4 μm sections. After antigen retrieval and incubation in 3% H 2 O 2 , the sections were incubated with the primary antibodies: anti-HDAC2 (1:200, ab32117, Abcam, Cambridge, UK), anti-p-CREB (1:100, ab32096, Abcam) or anti-NR2B (1:250, ab65783, Abcam) dissolved in 1% bovine serum albumin (BSA) at 4°C overnight. Then, the sections were exposed to the green fluorescent-conjugated secondary antibody (1:250, TransGen Biotech, Beijing, China) for 1 hr and DAPI stained for 5 to 10 min. Finally, the sections were viewed immediately using an inverted fluorescence microscope (200X) (Olympus, Japan). The photos were taken and the fluorescent densities of HDAC2, p-CREB and NR2B were detected using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD, USA). The images were converted to black and white. After intensity calibration, the pyramidal cell layer of hippocampal CA1 area was chosen, then the sum of area and the integrated optical density (IOD) were measured. The IOD /Area was calculated to determine the protein expression levels.
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