The overall goal of this procedure is to label proteins on the surface of living neurons and to subsequently distinguish surface protein from internalized proteins using differential secondary antibody labeling. To do this, primary neurons cultured on cover glass are incubated with an antibody directed against a surface epitope and incubated to allow for internalization. After the incubation, a fluorescently labeled secondary antibody is applied to identify surface exposed proteins.
Then excess unlabeled secondary antibody is applied to block any remaining surface protein primary antibody complexes. The cultured neurons are then perme and a secondary antibody of a different color is applied to detect internalized protein. Ultimately, results can be obtained that show the relative levels of internalized versus surface protein through immunofluorescence microscopy.
Following this procedure, a time course of source surface protein internalization can be obtained yielding information about protein trafficking rates under basil and stimulated conditions. Begin this procedure by preparing cover slips for cell culture wash polylysine coated 18 millimeter bolic glass cover slips prepared a day earlier by dipping them into three separate rinsing dishes containing PBS Add 500 microliters of laminate solution to the wells of a 12 well plate, and then using forceps, place the cover slips into each. Well press down on each cover slip to make sure it is sitting flat in the base of the well incubate for two hours at 37 degrees Celsius, this protocol can be performed using either E 15 mouse cortices or E 18 rat hippo Campi.
In this demonstration, mouse cortices are used. Place the cortices from half a litter or one litter of embryos on ice in a one milliliter einor containing PBS with calcium and magnesium and place on ice. Next, using a one milliliter micro pipette, remove the PBS from the cortical tissue.
Then add 500 microliters of a Pappa DNA's one solution to cortical tissue from half a litter of embryos. Incubate at 37 degrees Celsius for 15 to 20 minutes. Twice during the incubation period, gently flick the tube to mix the contents after the incubation, use a flame polished siliconized paster pipette to gently rerate the tissue 10 to 15 times until cell dispersion is obtained.
Few, if any chunks of non dissociated tissue should remain, avoid the generation of bubbles. Carefully layer the dissociated cell suspension over a three milliliter cushion of 4%BS, A in Hank's BSS with additives. Then in a benchtop centrifuge with a swing out rotor centrifuge at 100 times G for seven minutes.
After the spin, carefully remove the supernatant taking care not to aspirate the cell pellet. Then add one milliliter of complete neuro basal medium, gently pipette up and down to resuspend the pelleted cells. Next, use a hemo cytometer to determine the live cell concentration.
Only phase bright cells should be considered live immediately prior to plating. Aspirate the excess laminate serum solution from the cover slips in the wells and replace it with primary neuron culture medium. Then add 75, 000 to 100, 000 primary neurons to each well.
Culture the neurons for up to 21 days in neuro basal medium at 37 degrees Celsius, 5%carbon dioxide on day seven, and each week thereafter, add anti-mitotic fluoro, deoxy uridine uridine to prevent glial overgrowth and perform a half medium change. During the first week in culture, neurons developed dendritic arbors as shown in this image of a two day old culture. By the second to third week, they have matured and will be undergoing synaptogenesis.
An extensive network of projections is illustrated in this figure taken at 14 days in vitro to label surface exposed proteins of interest, add the primary antibody directly to the conditioned medium at the appropriate concentration. Each condition should be tested in triplicate Four controls, pre immune serum or antibodies that recognize intracellular regions of the protein that are not exposed during exocytosis can be used at the same dilution as the test primary antibody. Return the cells to the culture incubator for one, two, or four hours after the incubation aspirate the antibody containing medium, wash the wells once gently but rapidly with PBS.
Add room temperature by adding solution to the wells, then aspirating. Then add freshly prepared 4%para formaldehyde and incubate for five minutes. Add room temperature to fix the cells following fixation.
Remove the para formaldehyde solution from the wells and discard it in a liquid waste container in the fume hood. Then quickly rinse the cells three times with PBS as before. Leave the third wash in place so that the cover slip can be easily removed.
Next, use forceps to carefully remove the cover slips from the plate and place them cell side up on a sheet of param on the base or lid of a disposable culture plate. The remainder of the staining protocol will be performed on this surface gently pipette blocking solution of PBS 5%BSA onto the cells to make a rounded bubble shaped drop on the cover slip so that it does not dry out. Incubate for 30 minutes.
It's important to remember not to add detergent to the blocking solution. At this stage, the cells should not be permeated when only cell surface proteins are to be labeled. After blocking, aspirate the blocking solution, then to label surface exposed proteins, apply a fluorescently labeled secondary antibody, then incubate for two hours at room temperature following the incubation, aspirate the secondary antibody and wash the cover slips twice with PBS for five minutes each time to block any protein on the cell surface not bound by the labeled secondary antibody.
Incubate the nonpermeable neurons with a high concentration of unlabeled secondary antibody overnight. At room temperature. The overnight incubation is crucial.
Shorter incubation periods will not completely block any primary antibody that isn't fully bound by the labeled secondary antibody. The next day, wash the cover slips twice for five minutes each with PBS as before. Then postfix the cells with 4%para formaldehyde in phosphate buffer for five minutes at room temperature after removal of fixative.
Quickly rinse the cells twice with PBS. Next for intracellular labeling, perme and block the cells with PBS containing 5%BSA and 0.1%Triton X 100 at room temperature for 30 minutes. Following permeation, remove the blocking solution.
Taking care that the cover slips do not dry out. Add a differently tagged secondary antibody and incubate for two hours at room temperature. After removing the second secondary antibody, wash the cover slips three times for five minutes with PBS.
Then wash them briefly with deionized water mount. The cover slips on glass slides with an aqueous mounting medium containing an anti fade agent and allow them to dry store the slides in the dark at four degrees Celsius for optimal preservation of the fluorescent signal. Image the immuno stain cells on a confocal microscope using the appropriate excitation and emission filters.
Use the same imaging acquisition parameters for all replicates and conditions following imaging. Use standard image analysis software such as Fiji Image J or metamorph to determine the integrated density of standard regions of interest or the Punta attributes, such as number and size to determine whether seizure related gene six or says six was present on and internalized from the neuron surface. Dual color labeling of cultured rat hippocampal neurons was performed as described in this video.
In this image labeled cell surface protein is shown in cyan and internalized protein is shown in green. Double staining indicated by the arrowhead was only observed in a cell that appeared to be unhealthy to determine whether protein internalized during the antibody feeding incubation localized in endosomes neurons expressing the early recycling endosome marker transferrin mCherry were immuno stained after permeation as shown in this region of the dendritic arbor, there was extensive overlap of the punctate staining with transferrin confirming that the internalized protein localized to endosomes. The main advantage of this technique over existing methods is that a single antibody can be used to distinguish between cell surface and internalized protein pools.
