Hello,

A 10X Annexin V buffer contains 0.1M Hepes (pH 7.4), 1.4M NaCl, and 25 mM CaCl2 solution. Given that Annevin needs Ca ions to stably bind, I would not replace the buffer with plain PBS.

Even though I have not tried it, I would totally give it a try, since the pH and the osmolarity of the solution will not distort your cells or your antibodies.

I wish you best of luck,

George

Hello,

I prefer to stain my cells with antibodies in a buffer without calcium, which is contained in the Annexin staining buffer.

Hi Rohulla Vaseq,

If you don't want to use binding buffer you can use solutions contain CaCl2, even for fixation.

Of note, you have to fix your cells after staining with Annexin because fixation before staining is going to affect the results.

Best...

Annexin V is a calcium-dependent protein that binds phosphatidylserine (PS), which is exposed on the outer leaflet of the plasma membrane during apoptosis. Annexin buffer typically contains calcium ions to ensure proper binding of annexin V to PS.

In antibody staining, annexin buffer is not typically used. Antibody staining usually involves the use of specific buffers optimized for antibody-antigen interactions. These buffers may contain components such as blocking agents, detergents, or other additives to enhance the antibody binding and reduce nonspecific interactions.

If you want to perform annexin staining followed by antibody staining in two separate steps, it is generally not necessary to use annexin buffer for the antibody staining step. The calcium ions provided in the annexin buffer during the first step of annexin staining should have already facilitated the binding of annexin V to PS on the cell surface.

For the antibody staining step, you can use an appropriate antibody dilution buffer or staining buffer recommended for the specific antibodies you are using. These buffers are designed to optimize the antibody-antigen interactions and may differ from the annexin buffer in composition and purpose.