This protocol is significant because can be used to study the presence of glioma biomarker by a method that preserves the 3D structure of tumor neurospheres. The main advantage of this protocol is that the morphology of neurospheres is maintained compared to cytospin method in which cells are subjected to stressful manipulation and become flattened. This method can be applied to any other tissue culture systems in which cells are cultured in 3D and maintenance of the structure is important.
To begin, culture tumor neurospheres or NS in a T-25 flask until the cells are confluent. Elect the tumor NS in a 15 milliliter conical tube. Impellet the NS at 300 Gs for five minutes.
After this, re-suspend the pellet in one milliliter of 10%formalin and incubate the tube at room temperature for 10 minutes. Add five milliliters of HBSS to the re-suspended NS.Impellet the cells as previously described. Place the tube containing the pellet on ice.
Re-suspend the washed NS pellet in 500 microliters of two percent warm liquid agarose. And mix by a gentle pipetting or stirring. Place the agarose embedded NS on ice until the agarose polymerizes.
Then remove the piece of agarose, holding the NS.Place the piece of agarose into a cassette for tissue processing. Set a water bath to 42 degrees Celsius. And carefully insert the blade into the microtome.
Then place the paraffin block into the microtome. With one hand, press down on the lever that controls the thickness of each section located on the left hand side of the machine. Press the lever to the second stop to set the microtome to a 30 micrometer thickness.
Turn the coarse hand wheel to begin trimming the block. Once the surface of the sample is almost exposed, set the lever that controls thickness to five or 10 micrometers. Stop trimming when the surface of the sample is reached.
Fill an ice box with ice and just enough water to protect the paraffin block from directly touching the ice. Discard the old microtome blade and insert a new one for sectioning. Dry the paraffin block with gauze and place it on the microtome.
Turn the coarse hand wheel to begin sectioning the paraffin. Then use a damp paintbrush to transfer the paraffin ribbon to the 42 degree Celsius water bath. Using the curve of the forceps, place the forceps in between two paraffin sections and push the sections apart gently.
Use the damp paintbrush to push the separated sections onto positively charged adhesion microscope slides. First, melt the paraffin by incubating the slides in a metal rack at 60 degrees Celsius for 20 minutes. Then deparaffinize the slides via incubation in a re-hydration train at room temperature according to the text protocol.
Store the slides in tap water to prevent non-specific antibody binding. After this, incubate the slides in citrate buffer at 125 degrees Celsius for 30 seconds and 90 degrees Celsius for 10 seconds. Allow the slides to cool before rinsing them five times with distilled water.
Next, incubate the slides at room temperature in 0.3%hydrogen peroxide for 20 minutes. Then, wash the slides for five minutes, three times in washing buffer with gentle agitation. Incubate the slides overnight in a humidified chamber set to four degrees Celsius with diluted primary antibody.
After this, wash the slides three times for five minutes with gentle agitation. Next, incubate the slides in diluted biotinylated secondary antibody at room temperature for an hour. Wash the slides three times for five minutes in washing buffer with gentle agitation.
Then, incubate the slides in avidin-biotinylated horseradish peroxidase complex at room temperature in the dark for an hour. Repeat the wash as previously described. After this, incubate the slides with DAB hydrogen peroxide substrate brands for two to five minutes at room temperature.
Rinse the slides with tap water and dip the slides in hematoxylin solution to counterstain them. Then rinse the slides thoroughly in tap water until the water runs clear. Dehydrate the slides according to the text protocol.
Finally, coverslip the slides with a xylene-based mounting medium and allow them dry on a flat surface at room temperature. In this protocol, NS are fixed and embedded in agarose and paraffin. And then they are stained for specific glioma biomarkers.
The expression level of ATRX, a chaperone protein that mediates chromatin structure, is low. Ki67, a biomarker of cell proliferation, is positive in the glioma NS.Finally, the NS are also positive for OLIG2, a transcription factor that mediates oligodendrocyte differentiation. The most important thing to remember is to re-suspend the cells well in the warm agarose before transferring to ice, to ensure an even distribution of neurospheres.
