The overall goal of the following experiment is to identify regions of particular gene expression and to characterize the timing and levels of gene expression to contribute to the understanding of gene function. This is achieved by quickly harvesting and preparing the tissue to be used in the procedure to preserve the integrity of the tissue. As a second step, the tissue is treated to reduce the background binding of ribo probes.
Next, the ribo probes are hybridized to the tissue and the binding of the RIBA probe is visualized. Using x-ray film or light sensitive emulsion results are obtained that show the location and level of gene expression based on x-ray images or dark and brightfield microscopy and analysis. Using Image J software, There are about several critical steps that must be done well that many people make mistakes on and one is, is preventing background.
So there are a number of steps that can give you background such as poor sation or not hybridizing at the right temperature. Another is RNA contamination that will degrade your RNA probe. So you have to be careful about keeping everything RNAs free.
And the third is that the slides when they are dipped in the emulsion, you have to make sure you don't have any light leaks around in your dark room or exposure emulsion throughout all stages of that process, which can last for three weeks. So demonstrating this technique will be Hun Chen, who's a postdoc in my laboratory. After harvesting a fresh brain and rinsing in one times PBS embed the brain in OCT tissue tech media by immersing the brain in a mold containing the media at room temperature.
Ensure that the brain is orientated as needed for sectioning. Then after spraying the dry ice with ethanol, freeze the tissue by placing the block into the ethanol and dry ice mixture. Being careful not to get the ethanol inside the block using a cryostat slice the frozen tissue into 10 to 12 micron thick sections and mount onto super plus slides for brain and embryonic tissue.
The best cutting temperature is within the range of minus 18 degrees Celsius to minus 20 degrees Celsius. Store the sections in a slide box at minus 80 degrees Celsius. Transfer the slides from minus 80 degrees Celsius storage to a metal rack on dry ice.
Then while working in a certified fume hood, place the rack into a solution of 4%para formaldehyde that has been pre-warned to three to five degrees Celsius above room temperature. Incubate for five minutes at room temperature following incubation, remove the rack from the para formaldehyde and wash the slides by dipping the rack in PBS 15 times. Repeat this wash step twice using fresh PBS solution each time.
Next, immerse the slides in freshly prepared acetylation buffer for 10 minutes to reduce non-specific binding of the RIBA probe. Then wash the slides as before, but this time using two times SSPE as the wash solution. Dehydrate the tissue through a 70 95 and 100%ethanol series for two minutes in each solution.
Then dry the slides in the fume hood for at least 10 to 15 minutes. After synthesizing the RIBA probe, according to the instructions in the written protocol, calculate the required volume of RIBA probe and hybridization solution needed for all slides. Prewarm the Ribo probe hybridization mix to 65 degrees Celsius for five minutes.
To denature the Ribo probe pipette 100 microliters of the Ribo probe hybridization solution and align across the cover slip. Then use a second glass cover slip to spread the solution evenly over the tissue and cover slip. Place the slide horizontally and facing upright into a metal rack and once all slides are ready, immerse the rack slowly into a 65 degree Celsius oil bath for a minimum of four hours and a maximum of 16 hours.
The oil creates an airtight seal around the cover slips following the incubation tissue off any excess oil from the metal rack containing the slides. And then place the rack into a glass or metal tray containing chloroform in a fume hood. Repeat the wash with a second tray containing fresh chloroform.
Next, dip the rack into 0.1%betta me cap to ethanol plus two times SSPE solution to loosen the cover slips and dissolve excess hybridization solution. Then transfer the slides to a second solution of fresh 0.1%betta me cap TER ethanol plus two times SSPE solution. Transfer the uncovered slides into a fresh rack in a tray containing 0.1%beta macca TER ethanol in two times SSPE, incubating this solution for one hour at room temperature to remove excess unbound RNA probe.
Then transfer the rack of slides to prewarm two times SSPE plus 50%for main wide solution at 65 degrees Celsius containing beta ME capto ethanol to a final concentration of 0.1%and incubate at 65 degrees Celsius for one hour. After the incubation, discard this and all previous aqueous wash solutions as well as cover slips as radioactive waste. Next, wash the slides twice in pre-war 0.1 times SSPE has 65 degrees Celsius for 30 minutes each.
Then dehydrate the tissue through a 70%95%and 100%ethanol series for two minutes in each solution. Finally dry the slides in a fume hood for at least 30 minutes. First to determine whether a radioactive signal is present in the tissue.
Place the dried slides into a film cassette, working in a dark room using a safe light place x-ray film over the slides. Ensure that the slides are facing the emulsion side of the film and then close the cassette. Expose the slides to the film for one to seven days, depending on the expected abundance of the transcripts.
After the exposure is complete, develop the x-ray film in standard developer and fixer. The hybridization signal will appear as black areas on the film resulting from exposed silver grains in the emulsion next to determine cellular resolution and to see the signal on the tissue itself. The slides are dipped in photographic emulsion and then counters stained.
If the slides are to be counters stained with crestal violet, then the sections must be deli liquidized according to the instructions in the written protocol. At this point, working in the dark room, pour distilled water into a tube. Scoop out uc NTB emulsion and dilute the emulsion with distilled water to a one-to-one.
After melting the emulsion in a 42 degrees Celsius water bath for 20 to 30 minutes, transfer it into a glass dipping container in a 42 degrees Celsius water bath for 20 to 30 minutes. The level of the diluted emulsion should now cover all sections on a slide when the slide is dipped in it. If there are a lot of slides, extra emulsion may be needed.
Now dip the slides into the diluted emulsion in a 42 degree Celsius water bath, and then dry the dip slides in a closed light tight container overnight in the dark room or in an oven at 37 degrees Celsius for two to three hours with the lights off. After drying, transfer the slides to a slide box. Seal the edges with black electrical tape and then cover with aluminum foil.
Store the boxes at four degrees Celsius for several days to weeks when ready to develop, warm the slides to room temperature. Then while working in the dark room, remove the rack containing the slides from the boxes and develop them in the codec D 19 developer at 16 degrees Celsius for three to five minutes. Next, wash the developed slides in tap water at room temperature for one minute.
Then incubate the slides twice in fixing solution at 19 degrees Celsius for six minutes each the room lights can be turned on during the second fixer incubation. Now wash the slides in running water at room temperature for at least 30 minutes. Then using a razor blade, scrape the emulsion from the back of the slide while it is still wet.
Finally, counterstain with 0.3%crestal violet, then dehydrate mount slides according to the instructions in the written protocol if necessary. Further clean off any excess emulsion on the back of the slides by wetting with water and scraping with a razor blade. Rinse slides in 80%ethanol solution and wipe one to two times gently to get rid of debris and dust.
Take pictures under darkfield or brightfield illumination. The scraping and cleaning steps may have to be repeated two to three times in order to obtain good images without dust particles, which are easily seen in dark field under a dissecting microscope. The following images are examples of auto radiography of in situ hybridization from x-ray films.
This x-ray film image shows a sagittal whole head section of the zebra Finch songbird at embryonic day 10 that has been hybridized with an antisense ribo probe to Fox P one. The black exposed grains on the film show messenger, RNA expression as can be seen within the four brain Fox P one messenger, RNA is enriched in the meso alium stri aum and dorsal thalamus here. The x-ray film image shows a section from the same zebra finch songbird that has been hybridized with the 90 cents RIBA probe to Cuper.
TF two Co.TF two is enriched in the need of palam alium and more ventral thalamus. The following images are examples of auto radiography of in situ hybridization from a emulsion dip slides. This dark field image taken under a dissection microscope shows a sagittal whole head section of the zebra finch at post hatch day six that has been hybridized with an antisense RIBA probe to Fox P one.
The white exposed silver grains in the emulsion above the tissue shows messenger, RNA expression. The red color represents kressel violet stain. The scale bar represents one millimeter.
Here the image shows a section from the same zebra finch songbird that has been hybridized with an antisense ribo proof to coop TF two. The following images compare antisense and sense labeling that shows different patterns shown his auto radiography on x-ray films of Saul head slices taken from zebra finch at embryonic day 12. The dash line indicates the contour of the whole brain and the C marks the cerebellum.
It can be seen that the antisense strand of PAC six is expressed in the brain as especially in the ventricular zone. This adjacent section hybridized with the scent strand of pack six reveals no background expression throughout the embryo head, but apparent expression in the pigment layer of the retina as the antisense strand. The following images show in C two signals of gene expression and emulsion dipped slides taken from zebra finch brain during late embryonic stages under brightfield and darkfield views.
This brightfield image shows D one B expression at embryonic day 10. From a normal exposure to the emulsion, the label which appears black, can barely be seen at this magnification. This image shows an identical section and magnification as seen in the previous image, but switched to the dark field view showing labeling in white in the striatum and thalamus.
This bright field image shows slit three expression at embryonic day 12. After an over exposure to the emulsion, the black label can easily be seen. SC marks the position of the spinal cord and CB marks the position of the cerebellum.
Rostral is oriented to the left. The image shown here is the identical section and magnification is just seen but switched to dark field view showing labeling in white in the spinal cord that matches the bright field image. The following images show silver grain resolution at the cellular level.
Shown here is Fox P one messenger, RNA, labeling in the zebra finch forebrain silver grains appear as black spots above cell stained with crestal violet. This image shows high abundance expression over individual cells in the adult zebra finch meso alium. This image shows low abundance expression over individual cells in the adjacent alium of the same section here.
Hi Fox. P one messenger. RNA expression over cells is seen in the embryonic day 12 meso alium.
The embryonic cells seen here are smaller and more tightly packed compared with the adult cells seen in the previous two images. Finally, this image shows low abundance expression over cells in the adjacent alium of the same section. On following this procedure, other method like immunochemistry can be performed in order to answer additional questions like which cell type the target gene is expressed on.
After watching this video, you should have a good understanding of how to do the institute hybridization. Don't forget working with radiative reagents can be extremely dangerous and precautions such as personal protections should be always taken during this procedure.
