Immunofluorescence Lab Quiz

DAPI, blue

What fluorophore binds to DNA and what color does it dye the nucleus?

Fixation, paraformaldehyde cross-links proteins in the cell and freezes the proteins

What is the first step in indirect immunofluorescence (part 2 protein localization) and briefly describe this process

Permeabilization, opening up small holes in the membrane by adding a mild detergent (PBS) to allow antibodies to diffuse across the membrane

There is another step involved in the the fixation process involving the cell membrane, what is this step and what is the purpose?

Blocking, addition of a serum protein (PBTN) binds to inherently “sticky” proteins inside the cell that could bind to the primary antibody. A second antibody containing the fluorophore is added

What step occurs after permeabilization? Briefly describe this step

Green transmitted fluorophore binding to alpha-tubulin in the cytoskeleton to visualize the microtubules

Which fluorophore binded to the second antibody during indirect immunofluorescnece? Where does it localize?

tdTomato, red

What protein were the L929 cells fused with to identify the subcellular locations of the unknown proteins? What color did it turn?

plain visible light to observe specimens that are naturally colored or stained

Describe what brightfield microscopy is

use special filters to visualize certain structures (cell walls, membranes, nuclei, organelles(

We also used phase contrast light microscopy to view the VERY THIN elodea sample, describe this type of microscopy

Irritate a specimen with a specific wavelength of light to excite the electron. As the electron returns to a relaxed state, the excess energy transmits light at a longer wavelength

What is the basic function of a fluorescence microscope?

First barrier filter lets through only blue light with wavelengths between 450-490

The fluorophore is excited at a wavelength of 488 (Alexa 488, blue light). What happens as the light hits the first barrier filter?

Reflects light below 510 but transmits light above 510 from the objective lens

The fluorophore is excited at a wavelength of 488 (Alexa 488, blue light). After passing through the first barrier filter, what happens as the light hits the beam splitting (dichroic) mirror?

Cuts out unwanted fluorescent signals, passing the specific fluorophore color (green at a wavelength of 520).

The fluorophore is excited at a wavelength of 488 (Alexa 488, blue light). After passing through the dichroic mirror, what happens as the light hits the second barrier filter?

Translocation of proteins ATPase activity into the mitochondria

What is the function of TOMM 20?

Cytoplasm into the mitochondrial membrane

Where does TOMM 20 localize?

Packages DNA molecules into chromatin

What is the function of Histone 2A

Inside the entire nucleus

Where does Histone 2A localize?

Structural role in maintaining nuclear organization in the nuclear membrane. Maintains cell mobility and gene regulation

What is the function of Lamin A?

Nuclear membrane

Where does Lamin A localize?

Structural role in cytoskeleton organization. Regulates signaling pathways

What is the function of alpha actinin?

Along actin filament bundles in a web-like appearance

Where does alpha actinin localize?

rRNA processing during ribosomal biogenesis and catalyzes protein assembly

What is the function of fibrillarin?

Nucleolus

Where does fibrillarin localize?

Catalyzing transfer of galactose (carbohydrate) onto proteins and lipids

What is the function of Beta 1,4 galactosol-transferase?

Golgi complex

Where does Beta 1,4 galactosol-transferase localize?