The PHEM buffer is mainly used for tissues and cell cultures during immunocytochemical immunostaining studies.
Most antigens, especially intracellular antigens, exhibit better staining with the PHEM buffer compared to the PBS buffer. It also has a more limited impact on biochemical reactions and enzymes.
The use of PHEM also ensures excellent preservation of cellular ultrastructure, particularly microtubules.
Schliwa, Manfred et al. “Structural interaction of cytoskeletal components.” The Journal of Cell Biology 90.1 (1981): 222-235.
Jacqueline Montanaro: Enhanced ultrastructure of marine invertebrates using non-toxic buffers.
Storage: 4°C – 8°C
Immunofluorescence: Tips for Cell Immunostaining
Expert Advice on Immunostaining
By Nancy Kedersha PHEM Buffer
Researchers undertaking immunostaining face a series of technical obstacles that can make the difference between obtaining clear and informative images or chaotic disorder or even the absence of signal altogether…,” says Nancy Kedersha, a teaching professor in medicine at Harvard Med School and the director of confocal microscopy at Brigham and Women’s Hospital. “Worst of all, the outcome might seem very specific but be misleading or simply false.”
In this guest blog post, Nancy offers her expert advice on immunostaining and highlights factors to consider when planning your imaging experiments using cultured cells…
Find the right working environment…
(… AKA, optimize your buffers.)
Antibodies are optimized by the immune system to function in the sodium-rich environment of the blood – which is why phosphate-buffered saline (PBS) is generally the buffer of choice for cell surface staining. However, the intracellular milieu is dominated by potassium, and efforts must be made to recreate these conditions.
The buffer system used during incubations and washes after fixation (see below) and permeabilization can make a huge difference to the signal intensity. Buffers such as PHEM or CSK have been developed for use in permeabilized and unfixed cells in function-based assays; these buffers can be used on fixed and permeabilized cells instead of PBS and are particularly useful with cytoskeletal or cytoplasmic antigens.
In terms of signal intensity (based on my own experience), about 10% of cellular antigens show much better staining in PHEM buffer than in PBS, 50% show moderately better staining in PHEM buffer, and 30% show no difference. The remaining 10% stain much better in PBS! Therefore, I regularly try both PHEM and PBS when using an antibody for the first time. There is also a selection of other buffers that you might want to experiment with (see below).
PHEM Buffer: Pipes 60 mM, Hepes 25 mM, EGTA 10 mM, MgCl2 2 mM, pH 6.9: acronym for PIPES, HEPES, EGTA, and MgCl2