A Practical Approach, Second Edition=Ronald D. Ho.pdf

604 DEVELOPMENTAL REPRODUCTIVE TOXICOLOGY: A PRACTICAL APPROACH, SECOND EDITIONcan be performed with densitometry when the substrate is permanent (e.g., in our studies densitometrywas performed using avidin-biotin-peroxidase complex with diaminobenzidine). The sectionsare not counterstained with histological stains because staining interferes with the densitometricanalysis. For quantitative applications, extreme care must be exercised to expose all specimens toidentical solutions for precisely timed incubation periods, as well as to include appropriate controlsfor endogenous peroxidase and nonspecific binding of either primary or secondary antibodies. Also,substrate development should be monitored on test slides to determine the reaction time thatproduces a density allowing detection of either greater or lesser densities in the specimens. Theintensity of immunostaining can be ranked by the investigator based on viewing under the microscope(typically three levels are assigned: low = 1, medium = 2, high = 3). However, this methodshould be considered semiquantitative at best and susceptible to observer bias (the scorer shouldbe “blinded” to specimen identity).There are several computer software products available for densitometric analysis (e.g., Image-Pro Plus, NIH Image), and these can generate density values from images of immunostainedsections. Capture of images through a digital camera mounted on the microscope offers a mostconvenient method, although print or film images are also analyzed with these programs (generallythis requires scanning of the film or print to generate a digital file). In all of the densitometricmethods, it is critical to acquire images under tightly controlled, uniform illumination conditionsfor consistency across specimens. The darkest and lightest regions must not be “off scale” (notmaximal values). All images intended for densitometric analysis should be acquired with a monochrome(not color) camera, and sections should not be counterstained. The densitometric data arerelative values for a specific protein, and treatment effects are most appropriately expressed ascomparisons between the same cell type in the same region of treated and control sections. Suchdata may be statistically analyzed by ANOVA (analysis of variance), but consultation with astatistician is recommended because these data sets may require transformation or other approaches.B. In Situ HybridizationWith this technique, mRNA transcribed from the gene of interest can be localized on either sectionedtissue or in whole mount preparations. These protocols require preparation of antisense probes thathybridize to the sense strand (mRNA) in the tissue. Before this method can be used, it is thereforenecessary to have a gene sequence from which to synthesize the antisense probe. Through acomputer search of a gene database, it may be possible to determine if the gene of interest hasbeen sequenced and entered in the database. A literature search may identify a research group thathas the cDNA (complementary DNA) for the gene of interest, and most investigators will makethe material available on request after publication of its sequence.Alternatively, the American Type Culture Collection (ATCC) maintains an NIH repository ofhuman and mouse DNA probes and libraries. 18 cDNAs are placed in the repository by investigatorsand can be ordered from the catalog for a small handling fee. cDNAs from ATCC or private sourcesare not usually ready to use for making in situ hybridization probes. To prepare a quantity of thecDNA for use in probe preparation, it may be feasible to amplify the sequence with polymerasechain reaction (PCR). Alternatively, the cDNA can be cloned into a plasmid vector. Preparation ofRNA antisense–labeled probes for in situ hybridization will involve in vitro transcription of thecDNA sequence, which requires a promoter sequence for the polymerase (typically T3 or T7polymerase). Cloning or insertion of the cDNA into an appropriate vector and production of largequantities of that plasmid in bacterial culture is a standard, classical method. When received aspurified isolated cDNA or in an unsuitable vector, the cDNA must be cloned into a suitable vectorby standard protocols (as described in molecular biology reference texts). It is also possible to usesynthesized oligonucleotide probes that require no cloning.The choice of probe type (RNA, double strand DNA, single strand DNA, oligonucleotide),labeling method (in vitro transcription, end labeling, nick translation, random primer synthesis),© 2006 by Taylor & Francis Group, LLC