Rapid Tissue Clearing in Pre-warmed Xylene Without Compromising Staining Adequacy and Histoarchitecture
K. C. Onyegbula *
Department of Oral Pathology, Faculty of Dentistry, College of Medicine, University of Ibadan, Ibadan, Nigeria and Department of Medical Laboratory Science, School of Public and Allied Health, Babcock University, Ilishan Remo, Nigeria.
O. N. Afahaene
Department of Medical Laboratory Science, School of Public and Allied Health, Babcock University, Ilishan Remo, Nigeria.
S. T. Awolaja
Department of Medical Laboratory Science, School of Public and Allied Health, Babcock University, Ilishan Remo, Nigeria.
O. Makinde
Department of Medical Laboratory Science, School of Public and Allied Health, Babcock University, Ilishan Remo, Nigeria.
*Author to whom correspondence should be addressed.
Abstract
Aims: To determine the adequacy of nucleo-cytoplasmic staining characteristics and preservation of nucleo-cytoplasmic morphology of mouse liver tissue cleared in pre-warmed xylene at predetermined temperatures and durations using haematoxylin and eosin staining procedure.
Study Design: Tissues for clearing were first divided into 3 broad experimental groups (A, B and C) based on pre-determined tissue clearing temperature. Each broad group were further divided into 4 sub-groups (A1 to A4, B1 to B4 and C1 to C4) based on duration of tissue clearing.
Place and Duration of Study: Department of Medical Laboratory Science, Babcock University, Ilishan-Remo, Nigeria and Department of Oral Pathology, College of Medicine, University of Ibadan, Nigeria. January-June, 2015.
Methodology: Tissues assigned to sub-groups A1 to A4 were cleared in pre-warmed xylene at 25°C for 30, 45, 60 and 90 minutes respectively, while tissues assigned to sub-groups B1 to B4 were cleared in pre-warmed xylene at 30°C for 30, 45, 60 and 90 minutes respectively and tissues assigned to sub-groups C1 to C4 were cleared in pre-warmed xylene at 35°C for 30, 45, 60 and 90 minutes respectively. As a consequence of this procedure, adequacy of nucleo-cytoplasmic staining characteristics and preservation of nucleo-cytoplasmic morphology were subsequently determined.
Results: Adequacy of nuclear and cytoplasmic staining were both assessed using a 2-point grading scale of 0 being inadequate and 1 being adequate, while preservation of both nuclear and cytoplasmic morphology were also assessed using a 2-point grading scale of 0 being poorly preserved and 1 being well preserved. Adequate nuclear and cytoplasmic staining were observed in tissues cleared in pre-warmed xylene at 25°C for 45 and 60 minutes, at 30°C for 30 and 45 minutes, at 35°C for 30, 45, 60 and 90 minutes. Well preserved nuclear morphology were observed in tissues cleared in pre-warmed xylene at 25°C for 30, 45 and 60 minutes, at 30°C for 30 and 45 minutes and at 35°C for 30, 45, 60 and 90 minutes. However, only tissues cleared at 35°C for 30, 45 and 90 minutes exhibited well preserved cytoplasmic morphology.
Conclusion: Adequate nucleo-cytoplasmic staining and well preserved nucleo-cytoplasmic morphology of tissues for histopathologic demonstration may be achieved rapidly by clearing tissues in 35°C pre-warmed xylene for as short as 30 minutes.
Keywords: Pre-warmed, duration, clearing, staining, histoarchitecture