Immunocytochemistry – Lipids

Unlike immunocytochemistry (ICC) with proteins, ICC staining of lipids requires a great deal more planning and consideration. Lipids can reside in the membranes of multiple intracellular compartments, all of which will respond to permeabilization and fixation reagents differently. Here we outline optimized procedures for staining of the plasma membrane and Golgi apparatus with anti-lipid antibodies.

Protocol

This protocol is recommended as a general guide for immunostaining of cells with antibodies for detecting lipids of interest. Specific steps for staining of subcellular cellular compartments are also included. Full protocols for general, plasma membrane, and golgi staining are included in the file above. 

General lipid ICC

1. Fix cells by adding 4% paraformaldehyde to cells in media for 20 minutes at room temperature.
2. Wash three times with TBS.
3. Permeabilize the cells with 0.5% Saponin or 0.01% Digitonin at RT for 15 minutes.
4. Wash three times with TBS.
5. Block with 10% Goat Serum in TBS either overnight at 4 ºC or 30 minutes at 37 ºC.
6. Add the anti-lipid or anti-PIP antibody diluted in 10% Goat Serum in TBS to the concentration suggested on the technical data sheet. Incubate for 60 minutes at 37 ºC.
7. Remove the primary antibody solution and wash for 5-10 minutes in TBS-Goat Serum 1%. Repeat twice.
8. Dilute the secondary antibody in 10% Goat Serum in TBS to the concentration suggested on the technical data sheet. Add the secondary antibody solution and incubate for 45 minutes.
   -For fluorescently conjugated secondary antibodies, this step and all remaining steps until mounting should be
   performed covered or in the dark.
9. Remove the secondary antibody solution and wash for 5-10 minutes in TBS. Repeat twice.
10. Remove excess wash buffer by blotting the edge of the coverslip.
    -DAPI or alternative nucleic acid stains may also be applied at this time if they are not present in the mounting media. Excess nucleic acid stain should also be removed prior to mounting.
11. Add the mounting media to a microscope slide and mount and seal the coverslip. Repeat this for all stained samples and store at 4 ºC in the dark until imaging.

Plasma Membrane ICC

1. Remove culture media from cells and fix with 4% formaldehyde (FA) and 0.2% glutaraldehyde (GA) in PBS for 15 minutes at room temperature (RT).
   -Fixation may also be performed at 4 ºC for 3 hours.
   Steps 2-8 should be performed on ice or at 4 ºC using pre-chilled buffers and solutions.
2. Remove the fixation solution and rinse three times with 50 mM NH4Cl in PBS.
3. Block and permeabilize the cells with Buffer 1 (see recipe below) supplemented with 5% normal goat serum, 0.5% saponin,
   and 50 mM NH4Cl for 45 minutes.

Golgi Apparatus ICC

All steps should be performed at room temperature.

1. Remove culture media from cells and fix with 2% FA in PBS for 15 minutes.
2. Remove the fixation solution and rinse three times with 50 mM NH4Cl in PBS.
3. Permeabilize the cells with Buffer 1 supplemented with 20 μM digitonin for 5 minutes.

Buffers

These are suggested buffers for incubation and wash steps for Plasma Membrane and Golgi ICC. Further details can be found in the full protocol.

Notes

1. These protocols are recommended as starting points and are based on published data for lipid and phosphatidylinositol phosphate immunostaining. Users are encouraged to adjust the protocols as needed based on the provided references.
2. Variablity in staining patterns may be observed across different cell types owing to distribution of specific lipids and cellular lipid composition.
3. The stringency of the fixation conditions can be adjusted by increasing the crosslinker concentration, i.e. 2% FA –> 4% FA –> 4% FA + 0.2% GA, as well as the temperature.
4. Most common crosslinking reagents are unlikely to react wtih lipids directly and will instead react mainly with proteins. As such, performing staining at 4 ºC vs room temperature may affect residual lipid mobility after an initial fixation in some membranes.
5. Post-fixation of cells after antibody staining may also reduce residual lipid mobility.
6. In general, we do not  recommend the use of acetone or methanol as cellular fixatives as they are organic solvents and can solubilize and extract lipids.
7. If the use of acetone or methanol is required to visualize a protein of interest during co-immunostaining for a specific lipid, then we encourage the user to compare the staining pattern for a given lipid with an alternative fixation procedure.
8. If using the ‘general lipid staining ’ protocol, the choice of permeabilization reagent should be made with the target cellular compartment in mind: digitonin and saponin are selective for cholesterol and therefore require it for effective permeabilization. Triton X-100 and Tween20 will permeabilize all membranes but may also completely solubilize some membranes such as the Golgi and ER.
9. At sufficiently high concentrations, Saponin, Triton X-100, and Tween20 may lyse cells or extract lipids. We do not recommend exceeding 0.5% Saponin, 0.2% Triton X-100, or 0.1% Tween20

FAQ

Echelon Biosciences carries antibodies directed at specific lipids. These may be generally used in the same manner as protein antibodies but special considerations may apply depending on the assay and antibody.

References

1. Hammond, G.R., et al., PI4P and PI(4,5)P2 are essential but independent lipid determinants of membrane identity. Science, 2012. 337(6095): p. 727-30.
2. Hammond, G.R., G. Schiavo, and R.F. Irvine, Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P(2). Biochem J, 2009. 422(1): p. 23-35.
3. Hammond, G.R., et al., Elimination of plasma membrane phosphatidylinositol (4,5)-bisphosphate is required for exocytosis from mast cells. J Cell Sci, 2006. 119(Pt 10): p. 2084-94.