Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

1. Dissociated Hippocampal Neuron Culture

1. Prepare coverslips (borosilicate glass):
   1. Wash in 100% ethanol.
   2. Air dry under UV irradiation.
   3. Coat with Poly-D-lysine (0.5 mg/ml in 0.15 M borate buffer, overnight at 4 °C).
   4. On the next day (the day of culture) wash 3x in PBS then coat with laminin (2.5 μg/ml, natural mouse laminin) + 5% v/v heat-inactivated fetal calf serum (FCS) diluted in PBS for 2 hr at 37 °C.
2. Dissect embryonic day 18 (E18) rat hippocampi and collect into PBS containing calcium and magnesium, chilled on ice. NOTE: all experimental protocols involving animals were approved by the Animal Ethics Committee of the University of Melbourne.
3. Prepare the papain and DNase I solutions from the Papain Dissociation Kit according to the manufacturer’s instructions.
   1. Add 50 μl DNase I solution to 1 ml papain solution.
   2. NOTE: Once first prepared, the papain solution and the DNase I solutions may be separately dispensed into aliquots (0.5 ml and 25 μl aliquots for papain and DNase I, respectively) and stored at -20 °C until needed.
4. Remove as much PBS as possible and incubate hippocampi (from one litter of embryos) with 1 ml papain/DNase I solution at 37 °C for 15-20 min. Gently flick the tube to mix the contents twice during the incubation period.
5. Triturate the hippocampal tissue gently (avoiding the generation of bubbles) with a flame-polished siliconized Pasteur pipette 10-15x until cell dispersion is obtained and few, if any, chunks of undissociated tissue remain.
6. Carefully layer the dissociated cell suspension over a 3 ml cushion of 4% w/v bovine serum albumin (BSA) in Hanks Balanced Salt Solution plus additives (HBSS⁺; see Section 1.6.1).
   1. Prepare this step gradient solution by dissolving the BSA at room temperature without stirring in HBSS containing 2 mM CaCl₂, 1 mM MgSO₄, 4 mM NaHCO₃ and 40 mM glucose (HBSS⁺) then sterile filter and store at 4 °C.
7. Centrifuge, 100 x g, 7 min in a bench top centrifuge with a swing-out rotor.
8. Remove the supernatant being careful not to aspirate the cell pellet.
9. Resuspend the pelleted cells gently with a flame-polished (siliconized) Pasteur pipette (or a 1 ml blue pipette tip) in 1 ml complete Neurobasal medium (with 2% B27, 0.5 mM L-glutamine supplemented with 1% FCS).
10. Count two 10 μl aliquots using a hemocytometer (N.B. only count phase-bright cells as “live” cells).
11. Plate primary neurons on preprepared coated glass coverslips (0.75-1 x 10⁵/18 mm coverslip in 12-well plate). Immediately prior to plating, aspirate excess laminin/serum solution from coverslips and replace with primary neuron culture medium (see below). N.B. The required number of coverslips needs to be determined in advance as coverslip preparation is begun the day prior to culture (see step 1.1).
12. Culture rat primary E18 hippocampal neurons for up to 21 days in vitro (DIV) in Neurobasal medium, 2% B27, 0.5 mM L-glutamine supplemented with 1% FCS (heat inactivated). Perform a half medium change at 7 DIV and weekly thereafter. The anti-mitotic fluorodeoxyuridine/uridine is added at 7 DIV; 1/1,000 dilution of 10 mM stock of each nucleoside) to prevent glial overgrowth.

2. Antibody Incubation with Live Neurons

1. During the first week in culture, neurons are developing dendritic arbors and by week 2-3, neurons have matured sufficiently to be undergoing synaptogenesis^6,7.
   1. At selected experimental time point/s, apply the primary antibody to triplicate wells containing primary embryonic neurons on coverslips. Add an aliquot of the antibody directly into the culture medium of the primary neurons to the desired final dilution Note: In our example, a rabbit polyclonal antiserum raised against a recombinant secreted form of the protein was diluted 1/500 by adding 2 μl to 1 ml of culture medium in well; a centrifugation for 10 min, 13,000 x g, RT can be incorporated prior to taking an aliquot of antibody as this will remove any particulate matter and can help reduce background.
   2. For the preimmune serum controls, add an equivalent amount of preimmune serum (to the same final dilution). If no preimmune serum is available, add the equivalent volume of Neurobasal medium or PBS (no primary antibody control). Alternatively, if a suitable primary antibody is available that recognizes intracellular region/s of the protein, this antibody can be added to a control well in order to test specificity of the surface staining.
   3. Return the cells to the culture incubator for 1-4 hr (this incubation period may be determined empirically). In our experiments, the antibody was present for the entire period although the experimental design could incorporate a pulse of antibody followed by a further incubation period after wash-out (a pulse-chase experiment) to assess the time course of internalization.
      OPTIONAL STEP: This incubation can be carried out at room temperature or even on ice to slow the rate of basal protein internalization, if necessary. If performed in a non-CO₂ controlled environment, the medium should be changed to one that is not bicarbonate buffered before addition of the antibody/antiserum. CAUTION: Mature neuron cultures (>14 days, and particularly cultured mouse embryonic neurons) do not tolerate full medium changes well.
2. We have used incubation times for this primary antibody internalization step of 1 hr, 2 hr, and 4 hr with good results although the optimal time will depend on abundance of the protein of interest as well as the dynamics of protein trafficking in the cells under study.
   NOTE: The dual-color images shown in Representative Results were obtained with an incubation of 1 hr at 37 °C in a tissue culture incubator.
3. After incubation for the desired period, aspirate off the antibody-containing medium. Wash the wells gently but rapidly once with PBS at room temperature.

3. Secondary Antibody Application to Fixed, Unpermeabilized Cells

1. Fix neurons with 4% paraformaldehyde in phosphate buffer pH 7.2, 5 min at room temperature Note: for best results, use freshly prepared fixative; alternatively use fix that has been stored at -20 °C and completely thawed so that no precipitated paraformaldehyde is evident. CAUTION: Paraformaldehyde fixative should be prepared and handled in a fume hood.
2. Remove fix from wells (transfer to liquid waste container in fume hood) and rinse 3x with PBS.
   1. OPTIONAL STEP: If desired, to save on antibody reagents, the coverslips can be carefully removed from the 12-well plate with forceps and placed cell-side-up, onto a sheet of Parafilm laid on the base of a disposable culture plate.
   2. Solutions can then be pipetted gently onto the coverslips so that they are completely covered without the solution flooding over the edge of the coverslip onto the Parafilm.
   3. This technique is suitable for short-term incubations (1-2 hr) however longer incubations (e.g. overnight) should be carried out in a humidified chamber. Alternatively, the coverslips can be placed back into the wells of the 12-well plate for the overnight incubation and sufficient volume should be added to ensure that the coverslips do not dry out.
3. Block for 30 min at room temperature with 5% BSA in PBS (Note: Do not add detergent to the blocking solution at this stage as it is important that the cells are not permeabilized).
4. In order to label surface protein before proceeding with detection of internalized protein, apply the first fluorescently labeled 2° antibody of choice.
   Note: In the example described here, the primary antiserum was raised in rabbit (in-house antibody) to a recombinant secreted isoform⁴. Thus, for the first secondary antibody to detect the extracellular region of the transmembrane isoform on the surface of unpermeabilized neurons, we used donkey anti-rabbit Dylight 649 (1/200 diluted in PBS containing 5% BSA). It is recommended to centrifuge diluted secondary antibody solutions (10 min, 13,000 x g, RT) prior to use.
5. Incubate the coverslips for 2 hr at room temperature.
6. Wash coverslips (in wells), 2x 5 min with PBS.

4. Blocking with Excess Unlabeled 2° Antibody

1. Block the unpermeabilized neurons with a high concentration (>0.1 mg/ml) of unlabeled 2° antibody.
   1. The unlabeled 2° antibody should be raised against the species in which the primary antibody was raised (in this case rabbit) by incubation overnight at room temperature.
   2. For this protocol, AffiniPure F_ab fragment Goat anti-Rabbit IgG (H+L) was used at a concentration of 0.13 mg/ml.
      NOTE: The overnight incubation was found to be crucial as a shorter incubation period (2 hr) was insufficient for complete blocking of primary antibody that was not fully bound by the labeled secondary antibody.
2. Wash coverslips (in wells), 2x 5 min with PBS.
3. After this blocking step, post-fix the cells with 4% paraformaldehyde in phosphate buffer pH 7.2, 5 min at room temperature. Rinse with PBS (2x) after removal of fixative (transfer fixative to liquid waste container in fume hood).

5. Permeabilization and Application of the Second Fluorescently Conjugated 2° Antibody

1. Permeabilize and block the cells with 5% BSA in PBS containing 0.1% Triton-X-100 at room temperature for 30 min.
2. Remove the blocking solution (taking care that the coverslips do not dry out). Add the second fluorescently-conjugated 2° antibody tagged with a different fluorophore.
   1. NOTE: It must be possible to distinguish this fluorophore tag from the one previously used, depending on the available excitation/emission filters on the confocal microscope (see below).
   2. For the example presented in this protocol, an Alexa Fluor 488-conjugated donkey anti-rabbit 2° antibody was used (1/200 in PBS, 5% BSA and 0.1% Triton-X-100). Incubate 2 hr at room temperature then remove the 2° antibody solution.
3. Wash coverslips 3x 5 min with PBS and, finally, wash briefly with deionized water.

6. Mounting and Imaging

1. Mount coverslips on glass slides with an aqueous mounting medium containing antifade (e.g. VECTASHIELD) and allow to dry. Store in the dark at 4 °C for optimal preservation of fluorescent signal intensity.
2. Image immunostained cells on a confocal microscope.
   1. Appropriate excitation and emission filters for detection of the two fluorophore signals must be available.
   2. If different experimental conditions are to be compared (for example, internalization rates under depolarized^8,9 or control conditions), ensure that all replicate coverslips from the various conditions are imaged with the same image acquisition parameters. The integrated density of standard regions of interest or the puncta attributes (e.g. number, size) from the resulting images can then be measured using standard image analysis software (e.g. Fiji/ImageJ, Metamorph).