Alexa fluor 647 donkey anti rabbit igg h l

For detection of autophagy in hippocampal tissue, immunofluorescent analysis was conducted. Under deep anesthesia, mice were perfused with cold PBS, followed with 2% paraformaldehyde and 2.5% glutaraldehyde. Paraffin-embedded brain tissue was cut into 10-μm-thick coronal brain sections. Brain sections were treated with primary antibodies against LC3B (ab192890, Abcam, England) and NeuN (MAB377, Millipore, USA) overnight. Primary antibodies were cleared with PBS, and brain sections were intubated with secondary antibodies: Alexa Fluor^® 488 donkey anti-mouse IgG (H+L) (A21202, ThermoFisher, USA) and Alexa Fluor^® 647 donkey anti-rabbit IgG (H+L) (A31573, ThermoFisher, USA). DAPI was used to stain cellular nuclei. The immunoreactivity was detected with a fluorescence microscope (Axio imager Z2, Carl Zeiss, Germany) with three filters under high magnification (400×). Quantification the immunoreactivity of LC3B staining was performed in three microscopic fields (400×) each section in a blinded way.

Animals were transcardially perfused with 4% paraformaldehyde in 0.1 M PBS following flushing with prewarmed PBS. The cochleae of the guinea pigs were removed and immersed in 4% paraformaldehyde for 1 h at RT. The organ of Corti was carefully dissected and permeabilized with 5% horse serum in PBS supplemented with 0.3% Triton X-100 for 1 h. The samples were then incubated with anti-CtBP2 IgG1 (1:100; 612044, BD Biosciences, San Jose, CA, USA), anti-GluA2 IgG (1:1000; MAB397, Merck KGa, Darmstadt, Germany) and anti-myosin 7a (1:100; sc-74516, Santa Cruz Biotechnology Inc., Dallas, TX, USA) polyclonal antibodies with 3% horse serum in PBS supplemented with 0.3% Triton X-100 overnight at 37 °C. After three washes with PBS, the tissues were incubated with secondary antibodies against CtBP2 (Alexa Fluor™ 555 goat anti-mouse IgG1, 1:500, A21127, Thermo Fisher Scientific, Waltham, MA, USA), GluA2 (Alexa Fluor™ 488 goat anti-mouse IgG2a, 1:1000, A21131, Thermo Fisher Scientific, Waltham, MA, USA), and myosin 7a (Alexa Fluor™ 647 donkey anti-rabbit IgG (H+L), 1:500, A31573, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 37 °C. The samples were mounted with DAPI Fluoromount-G^® mounting medium (SouthernBiotech, Birmingham, AL, USA) and covered with a coverslip for analysis. Images were obtained using an LSM 880 Zeiss confocal microscope.

Rabbit anti-ABCB1 (1:100, 22336-1-AP, Proteintech) and rabbit anti-CX43 (1:100, 26980-1-AP, Proteintech) were used as primary antibodies to stain the cell suspensions, and Alexa Fluor 647 donkey anti-rabbit IgG (H + L) (1:200, A-31573, Thermo Fisher Scientific) was used as the secondary antibody. PE/Cy7-anti-mouse CD31 (1:100, 102418, BioLegend) and Alexa Fluor 700 anti-mouse CD45 antibodies (1:100, 147716, BioLegend) were used to stain the endothelial and immune cells. Dead cells in samples were excluded by propidium iodide (PI) staining in indicated experiments. Samples were analysed using Canto II with BD FACSDiva software v8.0.1, and the results were analysed using FlowJo v10 (BD Bioscience, USA). Gating strategies have been provided in the figures as well as in Supplementary Fig. 10.

Frozen liver sections (10µm) were incubated with rabbit anti-VCAM-1 (1:200) or rabbit anti-cleaved caspase-3 Asp175 (5A1E) (1:100, Cell Signalling Technology USA) and sheep anti-von Willebrand Factor (vWF; 1:100) (Abcam, MA, USA) overnight at 4°C. Alexa Fluor^® 647 Donkey anti-Rabbit IgG (H+L) and Alexa Fluor 488 Donkey anti-Sheep IgG (H+L) (1:900; Thermo Fisher Australia) were used as secondary antibodies. Images were captured using the Nikon TiE microscope (Monash Micro Imaging Platform).

The antibodies used in this work were: recombinant human CXCL13, human CXCL13 Quantikine ELISA Kit, mouse CXCL13 Quantikine ELISA Kit, mouse CXCL12 Quantikine ELISA Kit, mouse anti-human CXCR5-PE mAb, goat anti-human CXCL13 polyclonal Ab, goat anti-mouse CXCL13 polyclonal Ab (R&D, Minneapolis, MN), rat anti-mouse CD68-PE, rat anti-mouse CD45-APC, rat anti-mouse CD4-PerCP-CY5.5, rat anti-mouse CD19-FITC, rat anti-mouse CXCR5-PE/CY7 mAb (Biolegend, San Diego, CA), mouse anti-human CD68 mAb (Dako, Glostrup, Denmark), rabbit anti-mouse SPP1 polyclonal Ab (Proteintech, Chicago, IL), anti-TTF1 (Abcam, Cambridge, UK), rabbit anti-mouse E-Cadherin mAb, EMT Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA), rabbit anti-human AhR polyclonal Ab, goat anti-human Lamin B polyclonal Ab, mouse anti-human ɑ-tubulin mAb (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human β-actin antibody (Sigma, St. Louis, MO), Alexa Fluor 488 Donkey anti-Goat IgG (H+L), Alexa Fluor 555 Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 Donkey anti-Rabbit IgG (H+L) (Life Technology, Thermo Fisher Scientific, Basingstoke, UK), and mouse SPP1 ELISA Kit (Shanghai GenePharma, Shanghai, China). BaP and DEX were purchased from Sigma.

24 hours after the Aβ oligomer treatment, the hippocampal neurons were fixed with 4% paraformaldehyde (Sigma, USA). Immunostaining was then performed using a previously described standard protocol [43 (link)]. The primary antibodies anti-GluA1-NT (Abcam, Cambridge, MA, USA) and anti-PSD95 (Abcam) were used at a dilution of 1 : 200, and Alexa Fluor 555 donkey anti-goat IgG (H + L) and Alexa Fluor 647 donkey anti-rabbit IgG (H + L; Life Technologies, Gaithersburg, MD, USA) were used at a dilution of 1 : 800. After staining, the cells were mounted on glass slides using Fluoro-Gel II with DAPI (Electron Microscopy Sciences, Hatfield, PA, USA) and were imaged with a Carl Zeiss LSM 700 confocal microscope (Zeiss, Germany). Images were acquired with the same optical slice thickness for every channel using a 63x oil objective and a resolution of 1024 × 1024 pixels.