The overall goal of the following experiment is to visualize the distribution and stoichiometry of epidermal growth factor receptors or EGFRs in the plasma membrane of intact and hydrated cells with fluorescence and electron microscopy. This is achieved by first growing EGFR expressing cells such as cos seven on microchips with a 50 nanometer thin silicon nitride membrane spanning over a centrally located window area. Next, the cells are incubated with a biotinylated version of the epidermal growth factor that binds to the receptor and changes the confirmation of EGFR leading to dimerization and activation.
Next, the cells are fixed in order to immobilize the receptors and incubated with stripped Hagen conjugated quantum dots that bind to the EGF activated receptors and the cells are imaged. Finally, a second fixation with glutaraldehyde cross links the stripped habit in conjugated quantum dots with the EGFR and stabilizes the sample for electron microscopy. The results show a mission of red fluorescence through fluorescent quantum dot imaging and EGFR distribution and stoichiometric state or clustering via scanning transmission electron microscopy.
This method provides unique information about the stock geometry and distribution of membrane receptors in intact cells and can be used to obtain insight into the activation of the epidermal growth factor receptor in normal and in cancer cells. It can also be applied to study other membrane receptors such as ion channels To begin while working under a chemical hood. For the first preparation steps of silicon nitride membrane microchips and after submerging an acetone according to the text protocol, transfer the microchips directly into a beaker of ethanol and wait two minutes, remove the chips from the ethanol and place them onto a clean room tissue.
When the chips have dried, transfer them onto a glass slide previously placed into a Petri dish. Then place the cover on the dish. Next, place the glass slide with the microchips into a plasma cleaner.
Run a five minute standard cleaning program to render the surface of the silicon nitride membrane hydrophilic. Then under the laminar flow hood, transfer the microchips from the glass. Slide onto a clean room tissue before placing them into a well of a 24 well plate filled with poly L lysine solution or PLL incubate for five minutes.
Then one by one, transfer the microchips into a second well containing HPLC grade water before rinsing in a second well of water. Finally, place the microchips individually into DM filled wells of a 96 Well plate and place the plate in a carbon dioxide incubator using a COS seven cell subculture. Prepare a mono dispersed cell suspension with a concentration of approximately five times 10th.
The fifth cells per milliliter. Add one droplet of cell suspension to each microchip in the 96 wall plate and incubate room temperature for five minutes with an inverted microscope. Begin to examine the areas of the microchips to achieve an approximate density of 20 to 30 cos seven cells per 150 by 400 micrometer window.
When the cells have sufficiently adhered, carefully transfer the microchips with the cell spacing up into new wells containing dm. When sufficient cells remained adhered, transfer the microchips with the cells facing up into new wells containing serum free dmm. Afterwards, incubate the microchips at 37 degrees Celsius in a carbon dioxide incubator several hours to overnight for the following steps.
Using 96, well plate refill the wells with the necessary solutions and use one rower column per microchip. Rinse the microchips by placing them in a well with fresh B-S-A-G-E-L-P-B-S and incubate a 37 degree Celsius for five minutes. Transfer the microchips into 300 nano molar EGF biotin solution and place them back into the incubator for three minutes because the cells will begin to endo the EGF labeled egfr.
Quickly use PBS to rinse the microchips three times. Then use 0.1 molar cate buffer or CB to rinse the samples. Once next, transfer the microchips into 3%PFA and incubate for 10 minutes.
After rinsing once with CB and three times with PBS, transfer the chips into G-L-Y-P-B-S and incubate for two minutes. Then use PBS to rinse two times. Now, place the microchips in 10 NANOMOLAR S-T-R-Q-D for 10 minutes before rinsing four times with B-S-A-P-B-S.
Then submerge one microchip in a glass bottom 35 millimeter dish prefilled with room temperature B-S-A-P-B-S to image the cells with DIC and fluorescence microscopy. Use a 40 x air objective for overview images of cells labeled with QD 6 55. Use a filter cube that excites the quantum dots between 340 and 380 nanometers with emission above 420 nanometers.
Minimize light intensity to avoid two intense light that might damage the sample. Set the exposure time to at least 300 milliseconds so that the signals of all qds are captured as QD fluorescence blinks adjust the light intensity and the amplification to provide bright images without visible image noise while minimizing light intensity. After imaging, place the microchip back into a 96 wall plate with B-S-A-P-B-S.
Rinse once in cb, then transfer a 2%glutaraldehyde and incubate for 10 minutes after another. Wash and CB use B-S-A-P-B-S to wash the microchip three times store in B-S-A-P-B-S prefilled wells of a new 96 well plate for every microchip. Fill a row of four wells with HPLC grade water.
After setting up the ESEM according to the text protocol, load a sample into the pre-COOL ESEM STEM stage by taking the 96 WAT plate out of the cooling container and placing it close to the opened ESEM stage. Place a clean room tissue beside it. Quickly rinse a microchip by dipping it four times for a second each in a well filled with HPLC grade water.
Briefly blot the backside of the microchip on the tissue and place it into the pre cooled stage with three microliters of cooled HPLC grade water. Wet the surface of the sample and fix the sample in the stage. Then place three additional three microliter water droplets on the stage close to the sample and close the specimen chamber.
After identifying a cell of interest, according to the text protocol record a STEM overview image of the cell, move to a peripheral region of a cell where the cell border is visible and zoom into 50, 000 X magnification. Record images showing individual qds and use pixel dwell times of 20 to 30 microseconds shown here are QD 6 55 labeled membrane bound EGFR in intact, fully hydrated COS seven cells. The DIC image here illustrates the membrane topography of the cells and the corresponding fluorescence image shows distribution of EGFR over the entire surface of the cell after three minutes of EGF biotin incubation.
These low magnification ESEM STEM images reveal fine structures such as filopodia extending from the cell towards neighboring cells and some structures inside the thinner cell regions. Thick central cellular regions, including the nucleus, appear white because transmission through the sample is not possible for electrons of the used energy. High resolution.
ESEM STEM images of the thinner peripheral regions shows QD labels on a membrane fold diagonally crossing the image. This membrane structure has a higher EGFR density than the surrounding membrane regions at several locations, two labels were at close proximity. Two examples with distances of 20 and 24 nanometers are indicated with arrowheads.
These pairs of labels are interpreted as belonging to EGF Fers. This image was recorded of a region with a lower EGFR density and fluorescent signal. Nevertheless, EGFR was also found here in dimers and two clusters of 10 or 11 EEG were present.
Once mastered, the sample preparation can be done within two hours, much faster than commonly used techniques for electron microscopy. The subsequent essem STEM imaging can provide up to 100 high resolution images within half a day.
