Immunofluorescence Experiment

What is the purpose of fixing and immuno-staining cells?

To visualize the internal structure of cells, including microtubules, actin filaments, the Golgi, and DNA.

Why is it important to test antibodies at the start of the year?

Antibodies can weaken with age, and testing ensures they work well for experiments.

What is a monoclonal antibody?

An antibody solution with a single exact "flavor" of antibody, designed to target a specific epitope.

What is phalloidin, and how does it differ from antibodies?

toxin that binds and stabilizes actin filaments, directly fluoresces and doesn’t need a secondary antibody.

What does Alexa-488 do?

It is a fluorescent molecule excited by blue light (488 nm) that emits green light.

What is the purpose of fixation in the protocol?

To preserve the cell structure by cross-linking proteins, using methanol or paraformaldehyde.

What does PBS (phosphate-buffered saline) do during the protocol?

It removes excess fixative and maintains the cells in a stable ionic environment.

What is the function of PERM buffer?

To permeabilize the cell membrane, allowing antibodies to reach the cell interior.

What does BLOCK buffer contain, and why is it used?

Contains PBS, BSA, and TWEEN 20 to reduce nonspecific binding of antibodies.

Why are coverslips placed cell-side down during antibody incubation?

To maximize contact between the antibodies and the cell surface.

Why is it important to incubate in the dark?

To prevent photobleaching of fluorescent molecules.

What is DAPI, and what does it bind to?

A fluorescent stain that binds to AT-rich regions of DNA, fluorescing blue under UV light.

What does Rhodamine-phalloidin do?

It binds to actin filaments and fluoresces red, excited by green light.

What is the purpose of secondary antibodies in the protocol?

To amplify the signal from primary antibodies and add fluorescence for imaging.

How do you seal the coverslip after mounting?

Use anti-fade with DAPI and seal the edges with nail polish.

What are the three key solutions used in the protocol?

FIX (paraformaldehyde or methanol), PERM (PBS + Triton X-100), and BLOCK (PBS + BSA + TWEEN 20).

What is the function of Triton X-100 in PERM buffer?

To create holes in the membrane for antibody access to the cell interior.

Why is the block buffer used before antibody staining?

To prevent nonspecific binding and ensure specific staining of target proteins.

What storage temperatures are used for antibodies?

Alpha-tubulin and GM-130 are stored at 4°C and -20°C, respectively.

What are the advantages of fluorescently labeled phalloidin?

It binds actin filaments directly and does not require a secondary antibody.