TY - GEN
T1 - Development of immunohistochemical triple staining method for the evaluation of different types of cell death in coronary thrombus
AU - Pertiwi, K. R.
AU - de Boer, O. J.
AU - Gabriels, P.
AU - MacKaaij, C.
AU - van der Wal, A.
PY - 2019
Y1 - 2019
N2 - Immunohistochemistry is a powerful technique to identify the presence, distribution and extent of certain antigens at protein level in tissue specimen. Multiple immunostainings enable the detection, colocalization and comparison of several markers within one specimen. However, an optimization study is firstly needed to ensure the expression of multiple markers is clear and distinguishable. We formulated an optimized sequential triple immunostaining protocol which could identify the simultaneous presence of three types of cell death namely necrosis, apoptosis and etosis. These following antibodies were used: C-reactive protein (CRP, necrosis), (cleaved) Caspase-3 (Casp3, apoptosis) and citrullinated-Histone3 (CitH3, etosis). Several antigen retrieval methods, various concentration and order combinations of those antibodies as well as different combination color of chromogens were tested on coronary thrombus materials obtained from patients with acute myocardial infarction (AMI). The results showed that CRP (1:4000) visualized with 3,3'-diaminobenzidine (DAB) in brown, is better performed as the first staining, followed by CitH3 (1:8000) visualized with Perma Blue as the second staining and Casp3 (1:500) visualized with Perma Red as the last staining. In conclusion, we presented an immunohistochemical triple staining protocol to identify the comparative presence of different types of cell deaths: Necrosis, apoptosis and etosis in coronary thrombus specimens.
AB - Immunohistochemistry is a powerful technique to identify the presence, distribution and extent of certain antigens at protein level in tissue specimen. Multiple immunostainings enable the detection, colocalization and comparison of several markers within one specimen. However, an optimization study is firstly needed to ensure the expression of multiple markers is clear and distinguishable. We formulated an optimized sequential triple immunostaining protocol which could identify the simultaneous presence of three types of cell death namely necrosis, apoptosis and etosis. These following antibodies were used: C-reactive protein (CRP, necrosis), (cleaved) Caspase-3 (Casp3, apoptosis) and citrullinated-Histone3 (CitH3, etosis). Several antigen retrieval methods, various concentration and order combinations of those antibodies as well as different combination color of chromogens were tested on coronary thrombus materials obtained from patients with acute myocardial infarction (AMI). The results showed that CRP (1:4000) visualized with 3,3'-diaminobenzidine (DAB) in brown, is better performed as the first staining, followed by CitH3 (1:8000) visualized with Perma Blue as the second staining and Casp3 (1:500) visualized with Perma Red as the last staining. In conclusion, we presented an immunohistochemical triple staining protocol to identify the comparative presence of different types of cell deaths: Necrosis, apoptosis and etosis in coronary thrombus specimens.
UR - https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85073890726&origin=inward
U2 - https://doi.org/10.1088/1742-6596/1241/1/012013
DO - https://doi.org/10.1088/1742-6596/1241/1/012013
M3 - Conference contribution
VL - 1241
T3 - Journal of Physics: Conference Series
BT - International Seminar on Bioscience and Biological Education
PB - Institute of Physics Publishing
T2 - International Seminar on Bioscience and Biological Education 2018, ISBBE 2018
Y2 - 28 October 2018 through 31 October 2018
ER -