Alexa fluor 633 goat anti rabbit igg h l

Larvae were fixed in 4% PFA for 2 h at room temperature followed by incubation in MeOH overnight at −20 °C. Larvae were gradually re-hydrated at room temperature before washing thrice with 0.1% PBST for 5 min and then incubated in blocking solution for 2 h with gentle shaking. This was followed by overnight incubation with the primary antibody (Purified Rabbit Anti-active caspase 3 (BD 559565) 1:200 in Block solution) at +4 °C with shaking. The following day, larvae were washed 6 × 20 min with 0.1%PBT at room temp with shaking, followed by incubation for 2 h in secondary antibody (Alexa Fluor 633 goat anti-rabbit IgG (H + L) (Invitrogen A21071) in block solution). The larvae were washed 3 × 15 min in 0.1% PBT at room temperature. Following staining, larvae were stored in Citi Fluor at +4 °C before mounting for confocal imaging.

The expression of neural stem cell, neuronal and glial markers was performed by immunocytochemical analysis. The cells were fixed by adding 4% buffered formaldehyde solution containing 0.1% saponin. Primary antibodies to Nestin (R&D, 2 μg/ml), SOX2 (BD Biosciences, 5 μg/ml), βIII-tubulin (R&D, 2 μg/ml), MAP2 (Sigma-Aldrich, 5 μg/ml), GFAP (DAKO, 5 μg/ml), and NF-200 (Sigma-Aldrich, 5 μg/ml) were used together with Alexa Fluor 488 goat anti-mouse IgG (H + L) and Alexa Fluor 633 goat anti-rabbit IgG (H + L) secondary antibodies (all 1:400; Invitrogen, USA). The cell nuclei were labeled with Hoechst 33342 stain (Thermo Fisher Scientific). Immunofluorescence was analyzed using a Nikon A1 scanning laser confocal microscope (Nikon Co., Japan). All the staining studies were conducted in series, with 5 repeats in each series.

Mice were anesthetized with 125mg/kg Ketamine and 10mg/kg Xylazine cocktail intraperitoneal injection. The brain was fixed by the transcardial perfusion of 4% PFA/PBS, followed by overnight incubation in an additional 4% PFA/PBS. After removing the fixative, the brain was cryoprotected with a 10–30% sucrose solution. The section was made with 30μm slices. Blocking was achieved with 4% BSA in PBS and 0.3% Triton X-100. Primary antibodies and working dilution are the following: Mouse anti-β-Amyloid antibody (1:500, clone: 6E10, Biolegend), Rabbit anti-NeuN monoclonal antibody (clone: A60, Millipore), and Rabbit anti-Iba1 antibody (Wako). The tissue was then incubated with the following fluorescence-labeled secondary antibodies; Alexa Fluor 488 Goat anti-mouse IgG (H+L) (Invitrogen), Alexa Fluor 633 Goat anti-mouse IgG (H+L) (Invitrogen), Alexa Fluor 633 Goat anti-rabbit IgG (H+L) (Invitrogen). The core-dense amyloid plaques were stained with 0.5% Thioflavin S (Sigma, T1892). The nuclei were stained with Hoechst33342 dye. The images were taken on a Zeiss AiryScan 880 confocal microscope and further analyzed with ImageJ/Fiji^{50 (link)}. Five fields per section were randomly chosen in the cortical layer V and analyzed for quantification. The average value of 5 fields was presented for each mouse.

Immunohistochemical analysis was performed using thick vibratome sections, as well as paraffin sections, by means of both fluorescent and immunoperoxidase detection. The spinal cord and brain sections were stained using antibodies to Nestin (R&D), SOX2 (BD Biosciences), βIII-tubulin (R&D), MAP2 (Sigma-Aldrich), GFAP (DAKO), NF-200 (Sigma-Aldrich,) macroH2A.1 (ab183041, Abcam), human-specific anti-mitochondria antibody (ab92824, Abcam), and STEM-121 (Y40410, TakaraBio), anti- CD3, CD31 and CD68 (Roche Diagnostics) (all as 5 μg/ml solutions in PBS). In the case of immunofluorescence detection with the use of vibratome sections, Alexa Fluor 488 goat anti-mouse IgG (H + L) and Alexa Fluor 633 goat anti-rabbit IgG (H + L) (all dilutions 1:400; Invitrogen, USA) were used as secondary antibodies, the fluorescence was detected with a Nikon A1 laser scanning confocal microscope (Nikon Co., Japan). Immunoperoxidase staining was performed on thin paraffin sections with a Benchmark ULTRA immunostainer (Ventana, USA) utilizing the manufacturer-recommended detection system.