Quantifying Apoptosis in Liver Tissue

Liver tissue specimens were embedded in paraffin and sectioned at 5 μm for processing by the TUNEL method using a commercial kit, using DAB peroxidase substrate (Roche Molecular Biochemicals, Meylan, France) and counterstained with methyl green. Specimens were evaluated by microscopy at high power magnification ( × 100) in a blinded manner. A total of 30 random fields were counted for each TUNEL-stained tissue sample. TUNEL assays on primary hepatocytes were performed following exactly the same procedure as we previously described.^{43 (link), 44 (link)}

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Lebeaupin C., Proics E., de Bieville C.H., Rousseau D., Bonnafous S., Patouraux S., Adam G., Lavallard V.J., Rovere C., Le Thuc O., Saint-Paul M.C., Anty R., Schneck A.S., Iannelli A., Gugenheim J., Tran A., Gual P, & Bailly-Maitre B. (2015). ER stress induces NLRP3 inflammasome activation and hepatocyte death. Cell Death & Disease, 6(9), e1879-.

Publication 2015

DAB peroxidase substrate

Assays Embedded paraffin Hepatocytes Liver Methyl green Microscopy Peroxidase Stained tissue Tissue Tunel

Corresponding Organization :

Other organizations : Inserm, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, Archéologie et Histoire Ancienne : Méditerranée – Europe

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Variable analysis

independent variables

Tissue preparation (embedding in paraffin, sectioning at 5 μm)

dependent variables

TUNEL-positive cells (apoptotic cells)

control variables

Tissue sample (liver)
TUNEL method using a commercial kit
DAB peroxidase substrate
Methyl green counterstaining
High power magnification (× 100) microscopy
Blinded evaluation

controls

Positive control: TUNEL assays on primary hepatocytes, as previously described in the cited references (43, 44)
Negative control: Not explicitly mentioned

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