Goat alexa594 conjugated anti mouse igg

Tagged RAGE and AT1 / AT1mt / AT2 in genetically engineered CHO cells were detected using mouse anti-V5 (Nacalai, Japan) and rat anti-FLAG (Novus Biologicals, USA) antibodies, in combination with rabbit Alexa488-conjugated anti-rat IgG and goat Alexa594-conjugated anti-mouse IgG (Thermo Fisher Scientific, USA), respectively. 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, USA) was used to counterstain the nuclei. Images were acquired with a fluorescence microscope (BZ-X700, Keyence, Japan).

Four or seven coronal sections were selected from each mouse from the septal and hippocampal region with reference to Paxinos and Franklin’s The Mouse Brain in Stereotaxic Coordinates [69 ] (Supplementary Figure S3). To examine the immunoreactivity of Aβ, neuronal nuclei (NeuN), and synaptophysin (SYN), free-floating sections were incubated overnight at 4 °C with the mouse anti-4G8 antibody (1:2000; Cat.# 800701, BioLegend, San Diego, CA, USA), mouse anti-NeuN antibody (1:100; Cat.# MAB377, Merk KGaA, Darmstadt, Germany), or mouse anti-SYN antibody (1:500; Cat.# S5768, Sigma-Aldrich), respectively, in blocking solution (0.05% bovine serum albumin, 1.5% normal goat serum and 0.3% Triton X-100 in PBS) [70 (link)]. After washing three times for five minutes in PBS, the sections were incubated with goat Alexa 488-conjugated anti-mouse IgG (1:200; Cat.# A11001, Thermo Fisher Scientific Inc., Waltham, MA, USA) or goat Alexa 594-conjugated anti-mouse IgG (1:200; Cat.# A11005, Thermo Fisher Scientific Inc.) for 1 h at room temperature. The tissue sections were mounted on ProbeOn™ Plus Microscope Slides (Thermo Fisher Scientific Inc.) and coverslipped with Fluoroshield™ with DAPI (Sigma-Aldrich) to counterstain the nuclei.