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Due to the heterogeneity of Immunostaining, two blocks from each case were stained and scored combined.
A median of two representative blocks from each case was selected for immunohistochemical and chromogenic in situ hybridization (CISH) analysis.
The Vancouver Hospital ER laboratory retained single archival tumor blocks from each case in this patient cohort.
Four-micron sections (4 μm) of representative blocks from each case were deparaffinized, rehydrated, and treated with 0.3% H2O2 for 5 min in methanol to prevent endogenous peroxidase activity and were immunostained by the peroxidase method (Envision System, DAKO, Carpinteria, California, USA) according to the manufacturer's recommendations.
Four-micron sections (4 μm) of representative blocks from each case were deparaffinized, rehydrated, and treated with 0.3% H2O2 for 5 min in methanol to prevent endogenous peroxidase activity and were immunostained by the peroxidase method (Envision System, DAKO, Carpinteria, CA, USA).
In all, 3 μm of representative blocks from each case were deparaffinised in xylene, rehydrated, treated with 3%H2O22 in TBS for 5 min to block endogenous peroxidase activity, and subjected to heat-induced epitope retrieval in microwave oven using 10 m M citric acid at pH 6.0.
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Sections (4 μm thick) were cut from formalin-fixed, paraffin-embedded tissues derived from whole tissue sections from representative blocks for each case.
One representative paraffin block from each case was used for the study.
One formalin-fixed, paraffin-embedded tissue block from each case was retrieved from the archives of the Department of Pathology and Laboratory Medicine, Memorial University, upon approval from Eastern Health's Medical Advisory and Board of Trustees Research Proposal Approval Committee.
Immunohistochemistry was performed on serial sections from a representative paraffin block from each case using a standard ABC method and the signal visualised using diaminobenzidine.
One block from each case was selected to analyze the expression of PSA, PSAP and AR on serial sections by immunohistochemical methods.
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