TITLE: Double immunostaining for PIP2 and phosphorylated ERK
AUTHORS: Hiraku MIYAGI, Michio Hiroshima, Yasushi Sako

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>        Immunofluorescence microscopy is used to determine molecular localization and to measure fluorescence intensity of activity in cultured cells. Reagents for fixation and/or permeabilization must be suitable for the molecule of interest. We developed a procedure to immunostain phosphatidylinositol biphosphate (PIP</span><span style = "vertical-align:sub;">2</span><span>) and phosphorylated Extracellular signal-Regulated Kinase (ERK) simultaneously. PIP</span><span style = "vertical-align:sub;">2</span><span> is a phospholipid component of cell membranes. ERK is a mitogen-activated protein kinase (MAPK) that localized in the cytoplasm and nucleus after phosphorylation triggered by ligand activation.</span></div><div class = "text-block"><span>        Different compounds are used to permeabilize cells in order to stain for these two molecules. Digitonin is used for PIP</span><span style = "vertical-align:sub;">2</span><span> and Triton X-100 is used for phosphorylated ERK. However, since Triton X-100 can solubilize cell membrane lipids, it is not suitable for PIP</span><span style = "vertical-align:sub;">2</span><span> labeling. In order to confer resistance against Triton X-100, we devised a method to fix anti-PIP</span><span style = "vertical-align:sub;">2</span><span> antibody on the cell membrane antigen before permeabilization with Triton X-100. </span></div><div class = "text-block"><span>        We modified a conventional immunostaining method to include secondary fixation and permeabilization after anti-PIP</span><span style = "vertical-align:sub;">2</span><span> antibody labeling. The secondary fixation probably immobilizes antibodies bound to PIP</span><span style = "vertical-align:sub;">2</span><span> on the cell membrane, perhaps by cross-linking them to surrounding protein molecules. Whatever the mechanism, this fixation enables PIP</span><span style = "vertical-align:sub;">2</span><span>-antibody complexes to remain on the membrane, despite treatment with Triton X-100, which is used to visualize phosphorylated ERK.</span></div><div class = "text-block"><span>        Since PIP</span><span style = "vertical-align:sub;">2</span><span> and ERK are major cell signaling molecules in various cellular phenomena, simultaneous detection and visualization of their phosphorylation states by this method should be useful for many studies.</span></div></div>

[STEPS]
?. [Cell culture]
Cultivation of cells on cover slipsSee "Materials" to sterilize cover slips.Wash cells in a 60-mm dish with 3 mL of DPBS.Add 300 µL of Accumax (Funakoshi) to the dish.Incubate 10 min at 37° C in an incubator with 5% CO2.Suspend cells in 0.9 mL of DPBS containing 0.5 mM EDTA.Transfer this suspension to a 1.5 mL tube and count the number of cells using a hemocytometer.Dilute the cells to 2.5 x 104cells/mL in DMEM.Place a sterilized cover slip in each well of a covered 24-well plate as needed. Then transfer the diluted cell suspension to each of those wells (400 µL/well).Leave the plate on the bench for 10 min.Place the plate in an incubator at 37° C with 5% CO2 and cultivate for 12 hr.In order to starve the cells, replace the medium with 600 µL/well of culture medium without serum.
?. [Immunostaining]
Activation of cells with EGFRemove 300 µL of medium from each well, leaving 300 µL/well.Add 100 µL of EGF solution (400 ng/mL in DMEM, final concentration is 100 ng/mL) or add medium without ligand.Incubate for 5 min at RT.
?. [Immunostaining]
First fixation and permeabilizationReplace the medium with 400 µL of 3% PFA/NaF/Na3VO4.Incubate for 10 min at RT.Rinse x1 and Wash x3 with HBSS (see Materials)Replace HBSS with 400 µL of 50 ug/mL digitonin/3% PFA/NaF/Na3VO4.Incubate for 10 min at RT.Rinse x1 and Wash x3 with HBSS.
?. [Immunostaining]
First blockingRemove the buffer solution from the 24-well dish.Block cells for 30 min at RT with 300 µL of Blocking One-P (Nacalai).
?. [Immunostaining]
Labeling with anti-PIP2 antibodyTransfer cover slips from the 24-well plate to the humidified box, placing them on the parafilm. Excess solution on cover slips must be blotted before placing them on the parafilm.Put 40 µL of anti-PIP2 antibody solution in blocking One-P (1:600) on each cover slip.Incubate for 30 min at RT.Remove the antibody solution from cover slips in the humidified box and then immediately transfer them to wells of a covered 24-well plate to prevent drying.Rinse x1 and Wash x3 with HBSS.
?. [Immunostaining]
Second fixation and permeabilizationReplace the medium with 400 µL of 3% PFA/NaF/Na3VO4.Incubate for 10 min at RT.Rinse x1 and Wash x3 with HBSS (see Materials).Replace HBSS with 400 µL of 0.01% Triton X-100/3% PFA/NaF/Na3VO4.Incubate for 10 min at RT.Rinse x1 and Wash x3 with HBSS.
?. [Immunostaining]
Second blockingDiscard buffer from the 24-well dish.Block cells for 30 min at RT with 300 µL of Blocking One-P (Nacalai).
?. [Immunostaining]
Labeling with TRITC-conjugated antibodyTransfer cover slips from the 24-well plate to the humidified box. Excess solution on cover slips must be blotted before placing on the parafilm.Put 40 µL of TRITC-conjugated anti-mouse IgM antibody solution (1:2400) in Blocking One-P on each cover slip.Incubate for 30 min at RT.Remove the antibody solution from each cover slip in the humidified box and then immediately transfer them to wells of the covered 24-well plate to prevent drying.Rinse x1 and Wash x3 with HBSS.
?. [Immunostaining]
Labeling with fluorescent dye-conjugated ppERK antibodiesTransfer cover slips from the 24-well plate to the humified box. Excess solution on cover slips must be blotted before placing them on the parafilm.Put 40 µL of Alexa647-conjugated anti-ppERK solution (1:300) on each cover slip.Incubate for 30 min at RT.Remove the antibody solution from the cover slips and then immediately transfer them to a covered 24-well plate to prevent drying.Rinse x1 and Wash x3 with HBSS.
?. [Immunostaining]
MountingMount cover slips on glass slides with 4 uL of Abberior mount solid. Store at 4° C in the dark overnight.
?. [Observation and Results]
Observation and measurement of intensityFluorescent images of cells are captured using a fluorescence microscope. We use an Olympus IX-81 with an sCMOS camera, ORCA Flash4.0 (Hamamatsu) and a 20x objective lens, UPLSAPO 20x (Olympus). The microscope is controlled using Metamolph (Molecular Devices).Filter sets used to capture fluorescent signals are as follows:                U-MRFPHQ (Olympus) for TRITC                U-M41008HQ (Olympus) for Alexa647Fluorescence intensity is measured using ImageJ software (NIH). An example of a fluorescent image is shown in Fig. 1 and distribution of cellular fluorescence intensities is shown in Fig. 2.                U-MRFPHQ (Olympus) for TRITC                U-M41008HQ (Olympus) for Alexa647Fluorescence intensity is measured using ImageJ software (NIH). An example of a fluorescent image is shown in Fig. 1 and distribution of cellular fluorescence intensities is shown in Fig. 2.