The overall goal of this procedure is to use immunohistochemical staining techniques to evaluate the expression of B seven H one PD L one on paraffin sections of human pancreatic adenocarcinoma tissue. This is accomplished by first removing the paraffin from and rehydrating the sliced tissue sections. The second step of the procedure is antigen retrieval, which involves the use of a pressure cooker to stain the tissue.
An antibody staining procedure is followed by signal amplification and DAB color development steps. The final steps are to dehydrate and mount the tissue. Once complete.
Microscopy and image analysis can be used to visualize membranous and cytoplasmic B seven H one. This matter can help answer key questions in the study of a pancreatic endo carcinoma by giving us a better understanding of how B seven one BTL one contributes to the suppression of anti-tumor immunity in the tumor's microenvironment. The main advantage of this technique over existing methods, such as staining on cryo sections, is that it has been optimized for use of paraffin embedded tissue sections, which are often more easily available and more readily stored.
Reliable antibody staining on slides isn't possible without removing the paraffin first. So to prepare slides with paraffin embedded tissues, first bake the slides at 55 degrees Celsius for 20 minutes. Next, in a chemical hood, a series of seven chemical baths are used to remove the paraffin, first, immerse the slides in xylene for 10 minutes.
Next, immerse the slides in a second fresh xylene bath for 10 minutes. Third, immerse the slides in 100%ethanol for five minutes. Fourth, once again, immerse the slides in fresh, 100%ethanol for five minutes.
Fifth, immerse the slides in 95%ethanol for five minutes. Sixth, immerse the slides in 80%ethanol for five minutes. Finally, wash the slides clean with deionized water for five minutes.
Once paraffin has been removed, it is next necessary to make the antigen available. First, immerse the slides in Tris EDTA buffer. Now heat the slides in a pressure cooker with a two step program.
First, heat them at 125 degrees Celsius for 30 seconds. Second, 90 degrees Celsius for 10 seconds. Then set the slides aside to cool to room temperature.
After allowing the slides to cool for an hour, wash the slides with TBST twice, five minutes per bath. The slides can now be stained to each slide. Begin by blot drying the TBST solution around the tissue, and then marking a circle around the tissue with a hydrophobic pen.
Throughout this procedure, avoid the drying of the tissue by keeping the slides in a humidified chamber. When not being bathed, place one drop of peroxidase block within the hydrophobic circle on the slide. Wait five minutes and rinse the peroxidase off with TBST.
Then bathe the slides in TBST for five minutes. Remove the slides from the solution and gently shake off the excess TBST. Now place one drop of a CE blocking buffer on each slide and incubate them at room temperature for 15 minutes.
Clean the slides with a rinse and a five minute bath in TBST. Next place one drop of Vidan solution on each slide and wait 15 minutes. Remove the AVADON with the same TBST rinse and bathe regime.
Now apply biotin. Wait five minutes and clean the slides with TBST again. Now incubate the slides with a dilution of mouse monoclonal antibody or a control antibody.
In TBST allow either antibody to conjugate at four degrees Celsius for 22 hours. The next day, wash the slides with TBST three times as before. Each wash includes A-T-B-S-T rinse and a five minute bath.
This triple wash regime is repeated several times going forward while washing off the primary antibody. Prepare the STR titin Bio ITIN complex solution. It should be prepared at least 30 minutes before it's use.
Now incubate the slides with biotin conjugated goat anti-US secondary antibody in a goat serum solution for 30 minutes. During this incubation, follow the DACO kit instructions to prepare the DAB substrate CHROMOGEN solution. The solution can be stored at four degrees in the dark for up to eight hours.
After the incubation, perform the TBST triple wash. Next place one drop of strippin biotin complex. On each slide, incubate the slides for 15 minutes and then wash the slides three times with TBST.
Next, apply a drop of amplification reagent on each slide. Wait four minutes and wash the slides three times with TBST. After the amplification reagent is washed off, add a drop of streptavidin HRP to each slide and incubate them for 15 minutes.
Perform another TBST triple wash before proceeding. Now comes the most sensitive step to the procedure. Add one drop of DAB substrate CHROMOGEN with substrate hydrogen peroxide to a slide and begin observing the color development, The DAB substrate.
CHROMOGEN development time is typically two minutes, but we routinely monitor the development under a microscope to ensure optimal staining. With minimum background with each round of staining, we include one slide from a previous batch and one tonsil slide as our positive controls. When complete, immediately wash the slide in water, not TBST.
Incubate the slide with one drop of hematin for 90 seconds. Immediately wash the slide with distilled deionized water followed by soapy tap water and then more DD water. This completes the staining procedure.
Once all of the slides have been stained, use an ethanol series to dehydrate the slides before mounting them.First. Immerse the slides in 80%ethanol for three minutes. Second, immerse the slides in 95%ethanol for three minutes.
For the third and fourth baths, immerse the slides in 100%ethanol for five minutes. Fifth and sixth, immerse the slides in xylene for five minutes. Finally, add one drop of per amount to each slide and gently apply a cover slip.
The slides are now ready for microscopy immunohistochemical staining with B seven H one demonstrated the heterogeneous expression of B seven H one in pancreatic adenocarcinoma. They're seen as brown with DAB color development. Some pancreatic adenocarcinoma cells have predominantly membranous expression of B seven H one, whereas others have either predominantly cytoplasmic expression of B seven H one or little expression of B seven H one normal pancreatic duct epithelium expressed little B seven H one staining with a mouse IgG one kappa isotope control antibody instead of the anti B seven H one antibody produced very little background staining Once mastered, this technique can be performed in seven hours, spread over two days with a 22 hour overnight incubation if performed properly After this development.
This technique paved the way for researchers in the field of oncology to explore B seven H one PD L one signaling in other malignancies.
