Hello and welcome to the Calor D-U-I-D-U staining protocol. Today our video will begin with step three rehydration, permeation, and antigen retrieval. Begin By loading your Slides into the carrier.
Once your slides are loaded, place them into a xylene bath for five minutes. This will be followed by a second xylene bath. Again for Five minutes.
Continue to rehydrate your slides by moving them into ethanol solution, starting with 100%solution and finishing at 50%You will then complete slide rehydration by placing your slides for five minutes into one XPBS to perme cell membranes. Immerse your slides in 0.2%Triton X solution for five Minutes. Prepare your slides for microwave tissue antigen retrieval by placing them into a glass speaker with sufficient volume of 0.01 molar sodium citrate buffer so that the solution covers five centimeters above the top of the slides.
To account for evaporative Loss, Microwave slides Until the solution is boiling. This should take about three to 10 minutes at full power. Check that slides are boiling.
Reduce microwave power between 10 and 80%and continue slowly boiling the slides for an additional 20 minutes. After boiling place the beaker containing the slides on a bench top to allow them to slowly cool for approximately two hours until they've reached room temperature. Confirm that slides are at room temperature.
Immerse the slides in 1.5 natural hydrochloric acid for 40 minutes at room temperature. Remove your slides from the hydrochloric acid and place them for five minutes into A one XPBS wash. This will be followed by a second one XPBS wash.
Remove your slides from the one XPBS access solution with a liquid blocking wax pen. Circle each tissue section and replace the slide back into the PBS. Make a humid incubation chamber by placing a wet paper towel at the bottom of an airtight container.
Make up blocking solution of 10%docking serum diluted in one XPBS. It's important to entirely cover each tissue section with a solution for tissue sessions that are typical about one by 1.5 centimeters. A volume of 50 microliters of blocking solution is Sufficient.
Prepare the primary IDU antibody by diluting mouse anti BRDU antisera in 5%donkey serum. Add a concentration of one to 250. Pipette the primary solution Onto each slide section.
Place your slides into a refrigerator at four degrees Celsius. For an overnight incubation, prepare a high stringency wash. TBST works well.
Orienting slides back to back. Place the slides in about 35 to 40 milliliters of TBST Solution. Place The tubes in a shaking bacterial culture incubator at 37 degrees Celsius.
Allow them to shake for 20 minutes at 2 25 RPM. Now load your slides into a carrier. Place your slides in one XPBS for a five minute PBS wash.
This will be by a second one XPBS wash for five minutes. Prepare the primary CLDU antibody solution by diluting rat anti BRDU in 5%donkey serum. Add a concentration of one to 250 tap slides on a paper towel and then resection with a wax pen.
Use a pipette to cover each tissue section with primary CLDU solution. Use about 50 microliters of solution to make sure that every tissue section is completely Covered. Place your slides in a four degrees Celsius refrigerator for an overnight incubation.
Place your slides in a carrier and then place them in a one XPBS wash for five Minutes. Place your slides into a second, one XPBS wash for five minutes. Prepare secondary antibodies by diluting CY two donkey anti RAD and PS five donkey anti muse antibodies in 5%Donkey serum DPI will also be used for Nuclear staining, completely Cover tissue sections with secondary antibodies.
50 microliters of solutions should be sufficient to completely cover your tissue. Place the rehydration chamber in a dark place at room temperature for a One hour incubation. Place your slides in a carrier and then place them in a one XPBS wash for five Minutes.
Place your slides in a second, one XPBS wash for five minutes. Tap off Excess PBS solution on a paper towel. Then take prolonged gold anti fade mounting media and place a small drop of it on each tissue section.
Take a cover slip and place it gently on top of your slide sections. Use the paper towel as a press to push out any excess air bubbles that may have been trapped between your slide sections and the cover slip. Finally, use a pipette tip to push out any excess air bubbles that may remain image Your tissue of interest using a fluorescent microscope equipped with a high energy, light source and multiple fluorescent channels To quantify results.
Begin by counting the number of Dabi positive cells. Count the number of CLDU positive cells using the SI two channel image. Count the number of IDU positive cells using the SCI five channel image.
Finally, Count double positively labeled cells by flipping between the SCI two and SCI five channel image file.
