TY - JOUR
T1 - Statistical methods for in situ hybridization
T2 - Identification of autoradiographically labelled cells and structures
AU - Wessendorf, M. W.
AU - Wang, H.
AU - Schnell, S. A.
PY - 2004/7
Y1 - 2004/7
N2 - In situ hybridization experiments frequently use autoradiography to identify labelled structures. Ideally, labelled cells will be overlain with a dense accumulation of particles, allowing one to discriminate them from unlabelled cells easily. However, if noise is high or the density of labelling is low, it can be difficult to distinguish bona fide labelling 'by eye'. In such situations, labelled cells could be overlooked. This paper evaluates two statistical solutions to this problem: (1) a parametric method proposed by Hashimoto and co-workers and (2) Wang & Wessendorf's non-parametric method using contingency testing (i.e. the chi-square or Fisher's exact tests). The Hashimoto method determines the mean and standard deviation of the density of background labelling, using sense-strand controls as the source of background levels. Cells labelled at densities greater than two standard deviations above the mean (P < 0.0455) are defined as significantly labelled. Contingency testing determines whether the grain density over a cell is significantly higher than that over the remainder of the image. When compared, the two methods gave similar results. The Hashimoto method may be more sensitive if most cells are labelled but contingency testing requires no assumptions about the uniformity of non-specific labelling.
AB - In situ hybridization experiments frequently use autoradiography to identify labelled structures. Ideally, labelled cells will be overlain with a dense accumulation of particles, allowing one to discriminate them from unlabelled cells easily. However, if noise is high or the density of labelling is low, it can be difficult to distinguish bona fide labelling 'by eye'. In such situations, labelled cells could be overlooked. This paper evaluates two statistical solutions to this problem: (1) a parametric method proposed by Hashimoto and co-workers and (2) Wang & Wessendorf's non-parametric method using contingency testing (i.e. the chi-square or Fisher's exact tests). The Hashimoto method determines the mean and standard deviation of the density of background labelling, using sense-strand controls as the source of background levels. Cells labelled at densities greater than two standard deviations above the mean (P < 0.0455) are defined as significantly labelled. Contingency testing determines whether the grain density over a cell is significantly higher than that over the remainder of the image. When compared, the two methods gave similar results. The Hashimoto method may be more sensitive if most cells are labelled but contingency testing requires no assumptions about the uniformity of non-specific labelling.
KW - 5-hydroxytryptamine
KW - Autoradiography
KW - Gamma-amino butyric acid
KW - Glutamic acid decarboxylase
KW - In situ hybridization
KW - Quantification
UR - http://www.scopus.com/inward/record.url?scp=2342549835&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=2342549835&partnerID=8YFLogxK
U2 - 10.1111/j.0022-2720.2004.01332.x
DO - 10.1111/j.0022-2720.2004.01332.x
M3 - Article
C2 - 15230875
AN - SCOPUS:2342549835
SN - 0022-2720
VL - 215
SP - 50
EP - 61
JO - Journal of Microscopy
JF - Journal of Microscopy
IS - 1
ER -