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2 protocols using anti caspase 6

1

Immunofluorescence of Apoptosis Markers

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Histological sections were treated with xylene, rehydrated through a graded ethanol series, and pre-treated in citrate buffer (pH = 6.0) 10 min at 98°C. The primary antibodies: cleaved caspase-3 (9664, Cell Signaling, USA), cleaved caspase-6 (9761, Cell Signaling, USA), cleaved caspase-7 (9491S, Cell Signaling, USA), cleaved caspase-8 (8592, Cell Signaling, USA), cleaved caspase-9 (9509, Cell Signaling, USA), and osteocalcin (ab93876, Abcam, UK), were diluted (anti-caspase-3: 1:50, anti-caspase-6: 1:50, anti-caspase-7: 1:50, anti-caspase-8: 1:200, anti-caspase-9: 1:50, anti-osteocalcin 1:100) and applied overnight at 4°C. Alexa Fluor® 488 (A11034, Thermo Fischer, USA) or Alexa Fluor® 594 (A11037, Thermo Fischer, USA) were diluted 1:200 and then applied for 40 min at room temperature (RT). Nuclei were visualised by ProLong Gold Antifade reagent with DAPI (Thermo Fischer, USA).
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2

Protein Expression Analysis Protocol

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Cells were lysed in RIPA lysis buffer containing protease inhibitor cocktail and then total protein was collected for protein quantification through Pierce Bicinchoninic acid (BCA) Protein detection kit (Bio-Rad Laboratories, Hercules, CA, USA). Afterwards, 20 µg of protein was subjected to 12% SDS-PAGE, and then proteins were shifted to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). After being sealed with 5% BSA, the membranes were processed with primary antibodies at 4 °C overnight. The primary antibodies including anti-GAPDH, anti-cyclin D1, anti-CDK4, anti-caspase-3, anti-cleaved caspase-3, anti-caspase-6, anti-cleaved caspase-6, anti-PARP, anti-RAB10, anti-E6, anti-E7, were all procured from Abcam (Cambridge, MA). After washing in TBST, membranes were probed with HRP-conjugated secondary antibodies (Abcam) for 2 h at room temperature, and then protein signals were analyzed via the enhanced chemiluminescence substrate system (ECL; Santa Cruz Biotechnology, Santa Cruz, CA).
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