In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The overall goal of this procedure is to localize bacteria within paraffin embedded tissues using DIG labeled 16 s ribosomal, RNA, targeting DNA probes. This is accomplished by first preparing the probe with PCR amplification and DIG labeling. The second step is to prepare the tissue sections to have more accessible target sites and less background binding.

Next, the prepared tissue sections are hybridized with the dig labeled probe. Finally, the hybridized probe is detected with alkaline phosphatase, labeled anti-D dig antibodies. Ultimately, this in C two hybridization method can show the presence of bacteria with paraffin embedded tissues.

Domain advantage of this detect over existing method like ING is that this method can detect bacteria SHS within the context of some histological structures. This method can provide insight into the pathogenesis, but will various inflammatory such as periodontitis and R planet. Visual demonstration of this method is critical as the tissue preparation and hybrid digestion steps are difficult to run because the tissue sections are easily damaged.

To make the U bacteria probe use a 70 base pair, DNA fragment as a template and two 15 base pair primers to amplify the probe by PCR. Collect the amplicon in buffer at a known concentration. Then label the probe with a dig DNA labeling and detection kit.

First mix three micrograms of the probe into 15 microliters of water. Denature the probe in the heat block for 10 minutes at 95 degrees Celsius. Then put the denatured probe on ice.

Next, add two microliters of 10 XH nucleotides, two microliters of DN NTPs, and one microliter of canal enzyme. Once mixed, let the probe incubate at 37 degrees Celsius for 24 to 48 hours later. Stop the reaction by heating the mixture to 65 degrees Celsius.

Then measure the DNA concentration of the labeled probe at 260 nanometers and adjust a concentration to 100 nanograms per microliter. Using the kit, check the probes sensitivity first onto a nylon membrane load one microliter of serially diluted positive control. Probe and dig labeled probe.

Let the membrane dry for five minutes. Once dried, briefly dip the membrane into two XSSC and then transfer it to an 80 degree Celsius incubator for an hour. To immobilize the DNA after the incubation, briefly soak the membrane in maleic acid buffer, then transfer it to blocking buffer concentrated with AP conjugated antibodies at a one to 5, 000 dilution after half an hour.

Wash the membrane in MABT. Then mix 40 microliters of N-B-T-B-C-I-P into two milliliters of detection buffer. And after the bath, add this mixture to the membrane for one minute.

After removing the paraffin and rehydrating the tissue sections as described in the text protocol, prepare them for in C two hybridization first immerse the slides in 0.1 normal hydrochloric acid for 20 minutes. After the acid bath, wash the slides off with DEPC treated water for 30 seconds. Then using a wax pen a outline the specimens on each slide.

Next, add 50 to 400 microliters of proteinase K solution to the specimens, depending on their size. Allow the enzyme to work for 30 minutes at 37 degrees Celsius, and then wash the slides in DEPC treated one XPBS for one minute. Now transfer the slides to 4%paraform aldehyde in DEPC treated PBS for 10 minutes.

Wash off the fixative with PBS as before. Then bathe the slides in 0.1 molar triethanolamine hydrochloride at pH eight with 0.5%acetic and hydride after 20 minutes, wash the slides off with clean water as before. Now start the in C two by first immersing the slides in two XSSC for 20 minutes.

Meanwhile, dilute the labeled probe to one nanogram per microliter in hybridization buffer for a negative control. Use 10 times the concentration of non labeled probe probe. Then denature the probes by heating them at 90 degrees Celsius for 10 minutes, followed by quickly chilling them on ice.

Next, apply the same volume of probe dilution to each slide as proteinase K that had been previously applied. Then carefully cover slip and seal sections using nail polish. Now, heat the slides on a PCR machine block at 90 degrees Celsius for 10 minutes and transfer them to a 45 degrees Celsius humidified incubator overnight the next day.

Cool the slides down at four degrees Celsius for half an hour, and then carefully remove the cover slips. The tissues are fragile. Lastly, pass the slides through a series of SSC buffer at room temperature.

First, wash the NC two hybridized slides for five minutes in MABT. Then apply 50 to 400 microliters of blocking solution in 1%tween 20 for 20 minutes. After the block incubation apply 50 to 400 microliters of one to 1000 anti-D dig alkaline phosphatase antibody and blocking solution.

Let it bind for 90 minutes at 37 degrees Celsius after the primary has been applied. Wash the slides in MABT for 10 minutes. Then for five minutes, put the slides in detection buffer with 1%tween 20.

Now apply 50 to 400 microliters of one millimolar ole in detection buffer solution to each slide, and incubate them for five minutes to inactivate the endogenous alkaline phosphatase. Next, add 50 to 400 microliters of N-B-T-B-C-I-P diluted in detection. Buffer at one to 100 to each slide, and let the slides incubate for two to three hours in a humidified chamber based on experience.

Rinse the slides with DI water, then apply methyl green at 0.05%weight by volume and let the counter stain work on the tissue for 10 minutes. At 37 degrees Celsius. Wash the slides with DI water again, and then dehydrate them with 100%ethanol, followed by a dip in xylene.

Finally, apply 80 microliters of mounting media and cover slip. The slides after probes were dig labeled. Their sensitivity was compared to dilution of positive control probe.

The 3 43 base pair p gingivalis specific probe was 25 times more sensitive than the engineered 70 base pair bacteria probe. The two probes were then applied to gingival tissues from patients with chronic periodontitis at high magnification. The various shapes of bacteria were discernible using the bacteria probe.

While the p gingivalis specific probe only detected rod-shaped bacteria, a tight junction disrupting chemical DSS was applied onto the gingival mucosa of mice. Tissue samples from these mice were used to compare the probes in a different context. Here, the U bacterial probe was able to detect the bacteria the other could not.

The U bacteria probe was next tested for its ability to detect other bacteria in lung tissue sections containing tumors. Bacteria were detected outside the tumor as well as within the tumor. In examining tissues from patients with oral lichen planus a disease of unknown etiology, the bacteria probe detected bacterial signals within the cells of the epithelial and lamina propria where inflammatory infiltration was observed.

Since its development, this technique has helped the researcher in the field of infectious disease to explore bacteria invasion into tissues in both humans and animals Following this procedure. Other methods like immunochemical detection growth to cell marker can be ED in order to answer additional questions like what kind of cells harbor the bacteria within tissues.