Biomedical Engineering Reference
In-Depth Information
Note
*1: If not going to stain with cresyl violet, then delipidization is not necessary. It
is necessary to delipidize before placing slides in emulsion as the lipids will
not be removed easily after covering with emulsion.
*2: At this step, make sure the condition of the dipped slides is completely dry.
*3: Keeping dipped slides at 4 °C is critical to avoid mold growing on the glass
slides during exposure of the S
35
signal to the emulsion.
*4: Signal from 1 day on X-ray fi lm is similar to 5 days under emulsion.
*5: The slide needs to be wet; otherwise the razor blade will scratch the slides.
*6: This step can be performed at RT. In the case, the incubation time should be
longer.
9.9
Concluding Remarks
For visualizing radioactive probe signal on brain sections, there are major two ways,
detection by X-ray fi lms and by silver grains.
Detection by X-ray fi lms provides a relatively quick result and analyses.
Furthermore, the X-ray fi lm data could be used for high-throughput quantitative
analysis. The X-ray fi lm also reveals broad anatomical resolution. It provides the
ability to easily compare expression patterns among different brain sections on the
same and different slides. For this purpose, it is very critical to pay close attention
to obtaining a high signal-to-background noise ratio on the X-ray fi lm. High back-
ground on X-ray fi lms is usually caused by using old developer or fi xer or with
probe problem. Poor quality of riboprobes also generates high background. In our
experience, probes generated with Sp6 RNA polymerase more than T7 and T3 RNA
polymerases have a tendency toward such problems. In such case, we recommend
to increase enzyme units for RNA synthesis. The oil creates an instant seal, allowing
for rapid processing of many slides. Sometimes, mineral oil from the hybridization
step remains on the slides and tissue sections, which will cause spotted dark back-
ground. For quantitative analysis, we recommend not to use overexposed fi lms. It is
critical to maintain a signal intensity that correlates linearly with actual mRNA
expression levels. To show digital photo images of brain sections from X-ray fi lm,
there are two representative ways: regular bright-fi eld image (in this case, signal is
black in color: Fig.
9.7a
top low) and inverted black/white image (in this case, signal
is white color: Fig.
9.7a
2nd low). Inverted black/white images can be easily pro-
duced from regular bright-fi eld images using “inverted color function” in an image
software, such as Image J or Photoshop.
Detection by silver grains in the emulsion is produced by the pattern of decay
emission from the radioactive probe. The emulsion results show tissue morphology
and higher cellular resolution of gene expression. The emulsion contains light-
sensitive silver salts placed over the tissue. After exposure and developing the emul-
sion, exposed silver salts are converted to metallic silver grains. The metallic silver
grains block direct light through and appear as the black dots under bright-fi eld view.
