Alexa fluor 488 conjugated affinipure goat anti rabbit igg secondary antibody

Cell were seeded into 6-well dish at a density of 4 × 10⁵ cells per dish. After DA incubation, cells were fixed with 4% paraformaldehyde for 20 min and then incubated with 0.5% Triton X-100 for 10 min. PBS washing was conducted between each reaction to remove any residual solvent. Afterwards, fixed cells were incubated with 4% BSA at room temperature for 2 h and then with the appropriate primary antibodies at 4°C for overnight. After overnight incubation, cells were washed and then incubated with Alexa Fluor 488-conjugated affinipure goat anti-rabbit IgG secondary antibody (Jackson Immuno Research, West Grove, PA, USA) with light protection for 1 h. At the end of incubation, cells were observed under fluorescence microscopy equipped with filters for UV and Blue 488 nm.

Cells were seeded into 6-well dish at a density of 4 × 10⁵ cells per dish. After Polyphyllin G incubation, cells were fixed with 4% paraformaldehyde for 20 min and then incubated with 0.5% Triton X-100 for 10 min. PBS washing was conducted between each reaction to remove any residual solvent. Afterwards, fixed cells were incubated with 4% BSA at room temperature for 2 h and then with the appropriate primary antibodies at 4°C for overnight. After overnight incubation, cells were washed and then incubated with Alexa Fluor 488-conjugated affinipure goat anti-rabbit IgG secondary antibody (Jackson Immuno Research, West Grove, PA, USA) with light protection for 1 h. At the end of incubation, cells were observed under fluorescence microscopy equipped with filters for UV and Blue 488 nm.

Paraformaldehyde-fixed brains were sectioned coronally (at 20μm thickness) using a sliding microtome (CM1900; Leica, Germany). For immunohistochemistry (IHC), slices were blocked with phosphate-buffered saline (PBS) containing 10% fetal bovine serum (FBS) and 0.5% Triton X-100 for 1.5 hours, and incubated with anti-GFAP (Z0334; Dako Cytomation, Denmark), anti-Iba1 (019-19741; Wako, Japan), anti-YM1 (#01404, Stemcell Technologies, Canada) and anti-Calbindin-D_28K (CB38; Swant, Switzerland) antibodies at 4°C overnight. Slices were then incubated with Alexa Fluor 488-conjugated AffiniPure Goat anti-rabbit IgG secondary antibody (111–545–003; Jackson ImmunoResearch, USA) for 1.5 hours. For Thioflavin-S staining, slices were stained with 0.015% Thioflavin-S (T1892; Sigma, USA) for 15 minutes at room temperature. After mounting, slides were imaged using a Zeiss fluorescence microscope (Axio Observer A1; Zeiss, Germany).