The overall goal of this procedure is to prepare samples with low cell numbers to perform TEM analysis in an efficient and reproducible manner. This method can help to reveal all structures of real cells and a better physiological settings in the biomedical field. The main advantage of this technique is that both qualitative and quantitative results can be obtained from low cell numbers in reproducible manner.
To begin, use facts to sort 10, 000 hematopoietic stem cells, or HSC's into a 1.5 mL microcentrifuge tube. Containing 600 microliters of isocoves modified dulbecco medium with 10%FBS. Using a centrifuge with swing out buckets, spin down the sorted HSC's at 1, 000 times G for 10 minutes.
Use gentile aspiration to remove all but 200 microliters of the medium. To fix the cells add 0.2 mL of freshly prepared two X fixative solution, and incubate at room temperature for one hour. Next add 600 microliters of 0.1 molar cacodylate buffer to wash the cells.
Centrifuge the cells again, this time at 30 degrees Celsius. Then after centrifugation, and removing all but 200 microliters of buffer, add 200 microliters of two milligrams per milliliter of evans blue in cacodylate buffer, and incubate at room temperature for 20 minutes to stain the cells. Use 600 microliters of 0.1 molar cacodylate buffer to wash the cells three times.
Then re-suspend the cells in the remaining 200 microliters of buffer before transferring the cell's suspension into a 0.5 mL tube. At this step the pellet should be visible in the tube. With a table top centrifuge, spin the cells at 1, 000 times G for 10 minutes.
Then gently remove the buffer without disturbing the now visible tiny cell pellet, leaving 50 microliters of buffer in the tube. Prepare a 4%agarose solution, by adding 200 milligrams of low melting agarose to five milliliters of 0.1 molar cacodylate buffer in a 15 mL tube. Melt the agarose by transferring the tube to boiling water in a 100 mL glass bottle.
Add 200 microliters of the agarose solution to the tube of the cell suspension. Immediately centrifuge at 1, 000 times G, and 30 degrees Celsius for 10 minutes. After confirming that the cells pelleted into the semisolid agarose at the bottom of the tube, immediately transfer the tube to four degrees Celsius, or ice, for 20 minutes to solidify the agarose.
Using a 27 gauge needle, carefully transfer the solidified agarose, containing the cell pellet, to a 35 mL plastic petri dish containing 0.1 molar cacodylate buffer. With a scalpel trim the solidified agarose containing the cell pellet into one piece of about one to two millimeters. Then transfer it into a new 1.5 mL tube.
Add 600 microliters of 0.1 molar cacodylate buffer and wash the agarose cell pellet by using a pipette to add and remove the buffer. Remove the buffer from the agarose cell pellet and in a fume hood, while wearing protective clothing and gloves, add one milliliter of 1%osmium tetroxide solution. Incubate the cell pellet at four degrees Celsius for one hour.
After the incubation, wash the sample three times. Transfer it to a 20 mL glass scintillation vial with a cap. Next dehydrate the sample using an ethanol series.
Then infiltrate the sample in increasing concentrations of LX 112 resin in ethanol before incubating in 100%resin two times for 60 minutes each time. Using forceps, carefully transfer the sample to the bottom of a pyramid tip shaped rubber mold, and add pure resin on top of the sample to fill the mold. Incubate the sample at 60 degrees Celsius for 72 hours to polymerize the resin.
When the resin is polymerized, remove it from the molds by twisting the rubber mold. A small black cluster of cells should be visible in the polymerized resin pyramid. Use cyanoacrylate to attach the polymerized resin pyramid onto a mounting cylinder for sectioning.
After mounting the block onto an ultramicrotome use a razor blade and diamond knife to trim the pyramid block, making a short, wide trapezoidal shape with the top and the bottom of the block parallel to each other, and with evenly angled sides. Then use the diamond knife to trim the block around the cell pellet and remove the plastic sections around the cell pellet agarose piece. With the ultramicrotome cut one micrometer sections and use an eyelash tool and metal loop to move the sections from the water boat to a glass slide.
Then place the slides on a hot plate at 37 degrees Celsius to dry. Using a syringe with a 0.22 micrometer filter add a drop of teluatine blue solution to the sections and incubate for three minutes. Then with distilled water, rise the slides and let the sections dry.
After drying check the sections under a light microscope to identify the position of the cells. Next use a diamond knife to cut ultra thin sections. Then with an eyelash tool, move two to three sections onto 200 mesh grids from the water boat and place in a glass petri dish.
Transfer the dish with grids onto a hot plate at 30 degrees Celsius for 30 minutes to dry the sections. Add drops of 1%uranyl acetate, and incubate at room temperature for 10 minutes to stain the grids. Then, use distilled water to rinse six to eight times.
Stain with Reynolds lead citrate at room temperature for five minutes before rinsing six to eight times. Transfer the glass petri dish containing the grids to a hot plate at 37 degrees Celsius to dry the sections. Finally, analyze the sections with an electron microscope at 87 kilovolts.
Shown here at low magnification are semi-thin toluidine blue stained sections from blocks containing a tiny cell pellet that confirm a cluster of cells. This higher magnification image shows the intact morphology of the cells. This figure represents an over view field of the cell cluster from ultra thin sections that were stained with uranyl acetate and lead citrate and analyzed using electron microscopy.
Shown here is a single HSC, demonstrating that the morphology and ultra structure of the cells were well preserved. A higher magnification highly detailed electron micrograph of an individual HSC shows the intracellular ultra structures. In addition, high magnification images captured well preserved structure and integrity of subcellular organelles such as mitochondria.
Once mastered, this technique can be performed in five to six hours, and for advanced blue staining, and agarose embedding, and then normal TEM processing will take five to six days if it is performed properly. So after watching this video you sort of have a good understanding of how to prepare samples from real cell preparation for TEM Analysis.
