Anti-Beclin 1 antibody (ab62557)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Seed cells at 2 × 105/mL	- Primary Ab dilution-1:100 at 4°C overnight

Upstream tips
- Seed cells at 2 × 105/mL
Protocol tips
- Primary Ab dilution-1:100 at 4°C overnight

Publication protocol

Schwann cells were seeded at 2 × 105/mL on poly-l-lysine-coated coverslips. When they reached 70% confluence, the cells were starved overnight and observed in duplicate under the three conditions described above (control, or high glucose with or without quercetin). The cells were treated with 5% bovine serum albumin, followed by primary antibody (dilution 1:100; rabbit anti-Beclin-1 antibody and rabbit anti-LC3 A/B antibody; Abcam, Cambridge, UK) at 4°C overnight. PBS was used in place of primary antibody as the negative control.

Following PBS washes, the cells were incubated with secondary antibody (TRITC-conjugated Affinipure goat anti-rabbit IgG; Beijing Xiya Jinqiao Biotechnology Co., Ltd.) at 37°C for 20 minutes, and stained with fresh DAPI for 10 minutes. The fluorescent intensity of different groups of cells was analyzed using an Olympus FluoView FV 1000 (Olympus Corporation; excitation 364 nm, emission 488 nm for DAPI; excitation 547 nm, emission 620 nm for TRITC). Average absorbance values of each cell were measured by FV10-ASW 3.0 analysis software (Olympus Corporation). Five fields of view were randomly chosen from each group for analysis. The experiment was performed twice.

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Manufacturer protocol

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