The overall goal of this procedure is to label Edward Cella ilu bacteria and visualize them in situ you in catfish tissue sections using microscopy. This is accomplished by first challenging the fish with the bacteria. Next, the organs are sampled, paraffin embedded cut into tissue sections and dew waxed.
The bacteria are then labeled with primary and secondary antibodies. Finally, they're visualized using microscopy. Ultimately, results can be obtained that show the presence and distribution of the bacteria throughout the tissues using immunofluorescence microscopy.
Hello, my name is Simon Manto and the technique we are going to be presenting today was develop as part of my PhD thesis. The, the main advantage of this technique compared for example, to quantitative PCR bioluminescence, is that it allows to look at the bacteria in situ in the organ and investigate its location as well as its interaction with the host cells To challenge catfish. Tlu Punc Deus with bacteria begin by growing Edward Cella TLU overnight at 30 degrees Celsius in BHI broth.
Next, stop the water circulation in the fish tanks and add the bacterial broth at a concentration of one in 100. Allow the water to incubate for one hour, then restart the circulation to flush out the remaining bacteria. Sampling times and organs to sample depend on the objective of the study and what is known about the particular infection process analyzed.
In this study, we sampled the specified organs from six fish from each of the following time points. Once harvested, immediately submerged the organs in 1%formalin for two hours in histological cassettes. Then transfer the samples into 50%ethanol and keep them there until they're processed.
Embed the organs in paraffin and section To dewax. The samples after sectioning, immerse the slides in a staining trough filled with clear right for approximately five minutes to allow the wax to dissolve, lift the rack and drain off the excess wax solvent. Then immerse the slides in a second container of clear right for an additional five minutes.
Next, immerse the slides in pure ethanol twice. To remove the wax solvent transfer slides to 70%ethanol. And finally, run tap water to remove all traces of ethanol prior to immunohistochemistry.
Prepare the following buffers and solutions according to the written portion of this protocol. 0.9 x phosphate buffered saline blocking serum and 50 ER by carbonate buffered saline. Next, rehydrate the sections by immersing them in 0.9 XPBS for 10 to 15 minutes.
Incubate the slides for 30 minutes in blocking serum. Then incubate the sections in the EIC specific ED nine primary antibody for two hours in an opaque, moist chamber at room temperature. Rinse the sections four times in PBS for 10 minutes each.
Then incubate the sections for two hours and 50 microliters of the secondary antibody in an opaque, moist chamber at room temperature. Rinse the sections three times in bicarbonate buffered saline. Finally, rinse them briefly in deionized water to mount the slides.
Allow them to dry for a few minutes in the dark, then mount them using a mounting medium such as perma floor. Allow the mounting medium to dry overnight in a cool, dark environment. Finally, image the sections under a fluorescent microscope.
We use an Olympus BX 51 microscope equipped with a triple filter because the autofluorescence of fish tissues is so high, these tissues will appear orange under the tri filter. Conveniently providing a background for the bacteria against this background. The individual fitsy stained bacteria will appear as bright green dots and at 200 x, the rod shape will be recognizable.
After watching this video, you should have a good understanding as to how to label a do intersections using indirect to chemistry. Once mastered, this technique can be performed in five to six hours depending on how many sections you have to label. A lot of these steps, however, involve long incubation time so you can actually do other works.
In the meantime.
