NEUR 2035 Midterm - Lab 2

Question 1

Which of these flourophores is a fake product?
A. Excitation - blue // emission - green
B. Excitation - red // emission - green
C. Excitation - 490 nm // emission - 525 nm

Answer 1

B

Question 2

True or false:

FITC has purple to blue (or even greenish blue) as its excitation light. And blue is the best excitation light for FITC.

Answer 2

True

Question 3

What is the peak wavelength of FITC excitation light?
A. 358 nm
B. 490 nm
C. 525 nm
D. 490 mm

Answer 3

B

Question 4

True or false:

For our microscope, the light source for fluorescence comes from above the sample and the light source for bright field (the light microscope) comes from underneath the sample.

Answer 4

True

Question 5

Which is best practice when using a microscope?
A. Start with the 4x lens and randomly move the stage
B. Start with the 40x lens if you need to see detailed structures
C. Start with the 4x lens and the stage at the highest position

Answer 5

C

Question 6

True or false:

In the mouse brain, the majority of the fornix is located posterior to the majority part of the hippocampus.

Answer 6

False

Question 7

True or false:

When the accuracy of the volume is not a concern, we don’t have to use the second stop of the micropipette. And if generating a bubble is a concern, it is even better not to use second stop.

Answer 7

True

Question 8

True or false:

The first 3 wells of PBS are for getting rid off the storage medium from the brain slices.

Answer 8

True

Question 9

True or false:

It is better to transfer slices one by one as recommended by the lab manual.

Answer 9

True

Question 10

True or false:

The lens need to be covered here is needed in light microscopy. When we need the bright field light, it will focus the light on our sample so we can have a better image.

Answer 10

True

Question 11

True or false:

The blueish light (actually it is violet NOT blue) is the light that we need to excite DAPI (this part is true).
True or False: That light was generated from the light source underneath the stage.

Answer 11

False

Question 12

True or false:

The stage REALLY did not lower much before our image became clear. So it is a super smart idea to start with the stage in the highest position.

Answer 12

True

Question 13

True or false:

After DAPI staining, we can tell different layers of the meninges on a mouse brain slice under the 4x objective lens.

Answer 13

False

Question 14

If you find some DAPI-stained structures inside the lateral ventricle, is there any chance that they may be red blood cells?
A. Yes, because there are blood vessels in the lateral ventricle
B. No, because red blood cells don’t have nuclei

Answer 14

B

Question 15

True or false:

All images are made by tons of tiny dots. We call these dots pixels. Each pixel has 3 channels: red, blue and green. By changing the level of these 3 channels, we can manipulate colors of an image.

Answer 15

True

Question 16

True or false:

“white balance” can give us a starting place to adjust our image color, and “auto exposure” will give us a starting point to adjust our image brightness.

Answer 16

True

Question 17

True or false:

Fluorophore works by absorbing a higher energy light and emitting a lower energy light. The emitted light is the color that we will see.

Answer 17

True

Question 18

True or false:

Pigment works by absorbing a higher energy light and emitting a lower energy light. The emitted light is the color that we will see.

Answer 18

False

Question 19

True or false:

Pigment works by reflecting or scattering one wavelength of light and absorbing all other wavelengths of light. And the reflected or scattered light is the color of the pigment.

Answer 19

True

Question 20

True or false:

Flourophore works by reflecting or scattering one wavelength of light and absorbing all other wavelengths of light. And the reflected or scattered light is the color of the pigment.

Answer 20

False

Question 21

If you are using a 4x objective lense and a 10x eyepiece to view your sample, what is your magnification?
A. 100x
B. 40x
C. 10x
D. 4x

Answer 21

B

Question 22

If you need to see detailed structures in the nucleus of a cell from the CA1 region, which objective lens is the best choice?
A. 10x
B. 40x
C. 20x
D. 4x

Answer 22

B

Question 23

Which objective lens would you use to see more area of the CA1 region in one field?
A. 10x
B. 4x
C. 40x
D. 20x

Answer 23

B

Question 24

True or false:

The corpus callosum is comprised of axons.

Answer 24

True

Question 25

# True or false: In a neuron, the nucleus is located in the soma not the axon.

Answer 25

True

Question 26

# True or false: We can see a lot of DAPI stained cells within the corpus callosum.

Answer 26

True

Question 27

# True or false: The DAPI stained structures in the corpus callosum are contamination because there should not be nuclei in that region.

Answer 27

False | (what we are seeing are glial cell nuclei)

Question 28

Which statement is false? A. Corpus callosum is dorsal to caudoputamen B. Blue light is used as the excitation light for DAPI imaging C. We used PBS to rinse off the storage medium and extra DAPI from the brain slices

Answer 28

B

Question 29

If you have a brain slice from an organ donor who died from a brain tumor and you want to test if the neurons closer to the tumor have less dendrites than the neurons further away from the tumor, can you use DAPI to stain the brain slice to help you draw a correct conclusion?

Answer 29

No

Question 30

What solution do we put on the brain slices to protect the flourophore? A. water B. PBS C. mounting medium D. DAPI

Answer 30

C

Question 31

In the central nervous system, glial cells include... A. Microglia B. Astrocytes C. Oligodendrocytes D. Schwann cells E. Ependymal cells

Answer 31

A,B,C,E

Question 32

If your image looks very bright, which action(s) will help you to lower its brightness? A. Lower the exposure time B. Lower the gain C. Lower the number for the red channel

Answer 32

A and B

Question 33

The DAPI stained nuclei within the corpus callosum might belong to... A. Microglia B. Schwann cells C. Ependymal cells D. Astrocytes E. Oligodendrocytes F. Red blood cells

Answer 33

A, D, E

Question 34

If you are collecting images from a DAPI stained slide and need to leave for a short period, what should you do to prevent photo bleaching?

Answer 34

Close the curtain for the flourescent light path

Question 35

It takes about 30 minutes to do a DAPI stain. Is turning on the violet light at 100% intensity before you start the stain good practice?

Answer 35

No, that burns the light bulb for nothing

Question 36

# True or false: We can find anterior composure olfactory limb (aco) and hippocampus on the same coronal section in mouse brain sample.

Answer 36

False

Question 37

If we stain a brain slice with both NeuN and DAPI, what will be stained on on the slice? A. Astrocyte somas by NeuN B. Neuron somas by NeuN C All nuclei by DAPI

Answer 37

B and C

Question 38

# True or false: Among different antibodies, the variable regions are different. Thus they can bind to different epitopes. And the constant regions of antibody determine the mechanism used to destroy an antigen during an immune response.

Answer 38

True

Question 39

# True or false: When we call a stain “NeuN” stain, we mean NeuN is the epitope of our interest. With the help of antibodies we can stain all cells that contain NeuN.

Answer 39

True

Question 40

# True or false: Under the microscope: Neurons always appear orange-ish in color, Astrocytes always appear green in color, All nuclei always appear blue in color.

Answer 40

False

Question 41

Which set(s) of antibody will allow you to stain astrocyte on a mouse brain slice. A. Rabbit anti-mouse GFAP, donkey anti-rabbit Alexa-488 B. Chicken anti-mouse GFAP, goat anti-chicken Alexa-488 C. Chicken anti-mouse GFAP, donkey anti-rabbit Alexa-488 D. Chicken anti-mouse GFAP, goat anti-rabbit Alexa-488

Answer 41

A and B

Question 42

# True or false: The regions that protrude out of the 3D structure of a protein are called epitopes. And antibodies bind to epitopes.

Answer 42

True

Question 43

Secondary antibodies will bind to ____ in IHC staining. A. AT-rich regions of DNA B. DNA and RNA C. Epitope that primary antibodies bind to D. Primary antibodies

Answer 43

D

Question 44

# True or false: Different epitopes even from the same protein may have different amino acid sequences.

Answer 44

True

Question 45

# True or false: Antibodies are Y-shaped proteins, which means an antibody is made of amino acids and its 3D structure is a Y shape

Answer 45

True

Question 46

# True or false: After staining with antibodies, we can identify each individual proteins under a fluorescent microscope.

Answer 46

False

Question 47

# True or false: In the indirect immuno-fluorescence, the constant region of the primary antibody offers binding sites for the secondary antibody. The constant region of the secondary antibody offers space for fluorophore molecules to attach.

Answer 47

True

Question 48

GFAP is the name of an epitope, and it only exists in astrocytes. Alexa-488 is a fluorophore. Which set(s) of antibodies will allow you to stain astrocyte on a mouse brain slice. A. Chicken anti-mouse GFAP, goat anti-chicken Alexa-488 B. Chicken anti-mouse GFAP, goat anti-rabbit Alexa-488 C. Chicken anti-mouse GFAP, donkey anti-rabbit Alexa-488

Answer 48

A

Question 49

Which reagent is needed to fix brain samples? A. Triton-X100 B. Blocking reagent C. Antibodies D. Formalin

Answer 49

Formalin

Question 50

If a recent study just reports that certain mouse neurons do not contain NeuN protein. Do you think these neurons will be stained after NeuN IHC stain?

Answer 50

No - those neurons don't have epitopes for the primary antibody to bind to

Question 51

# True or false: The greener areas (most of them are line-shaped) contain GFAP protein. Primary antibody binds to GFAP, and fluorophore-labeled secondary antibody binds to primary antibody. Many fluorophore molecules accumulated there. Thus they are greener.

Answer 51

True

Question 52

# True or false: We cannot find NeuN protein in most parts of the neuron’s axon; However, we can find GFAP in both the astrocyte’s soma and processes.

Answer 52

True

Question 53

# True or false: In the caudoputamen, there are some regions that are white matter (mostly axons) and some regions that are grey matter (mostly soma); Astrocytes can be found in both the white and the grey matter regions of the caudoputamen.

Answer 53

True

Question 54

Which statement is wrong? A. Only a few neuron somas are found in cortical layer 1 B. There are more astrocytes in cortical layer 1 than in other layers C. Each orange spot in the NeuN stained image represents one NeuN protein.

Answer 54

C

Question 55

# True or false: Almost no neuronal somas can be found in the corpus callosum, but astrocytes are present in corpus callosum.

Answer 55

True

Question 56

# True or false: Layer 1 of the cortical layers has less neuron soma density than other cortical layers.

Answer 56

True

Question 57

# True or false: The caudoputamen has some astrocytes, but a lot of neuronal somas can be found there

Answer 57

True

Question 58

Which statement is correct about the experimental steps in this module? A. DAPI was introduced to the stain together with a secondary antibody B. We only stained the brain slices with NeuN and GFAP, not DAPI C. DAPI was introduced to the stain together with a primary antibody D. DAPI was introduced to the stain together with a primary and secondary antibody

Answer 58

A

Question 59

Which statement is correct about indirect immunoflourescence? A. We only label secondary antibodies with flourophore, not primary antibodies B. Neither primary nor secondary antibodies are labelled with flourophore. Instead we label antigens with flourophore at their epitopes. C. Both primary and secondary antibodies are labelled with flourophore to give a stronger signal

Answer 59

A

Question 60

When we make GFAP primary and secondary antibodies to stain a mouse brain slice, first we need to isolate ____ from the mouse. A. GFAP primary antibody B. GFAP antigen/epitope C. GFAP secondary antibody

Answer 60

B

Question 61

When we make GFAP primary and secondary antibodies to stain a mouse brain slice, we inject the GFAP antigen into a primary antibody animal (rabbit). Then we isolate the primary antibody from the... A. goat B. chicken C. rabbit D. mouse

Answer 61

C

Question 62

When we make GFAP primary and secondary antibodies, if the primary antibody came from a rabbit, what animal can we not use to make the secondary antibody. A. donkey B. dog C. goat D. rabbit E. horse

Answer 62

D

Question 63

Which structure is the most superficial in a brain with meninges? A. Hippocampus B. Meninges C. Glia limitans

Answer 63

B

Question 64

If we use rabbit anti-mouse antibody as our primary antibody to stain a mouse brain slice, which of these can we use as our secondary antibody? A. Goat anti-rabbit antibody B. Goat anti-chicken antibody C. Goat anti-donkey antibody

Answer 64

A

Question 65

Summarize the major IHC steps.

Answer 65

1. isolate epitope from animal to be studied 2. inject epitope into another species of animal (primary antibody animal) 3. kill animal and isolate primary antibody 4. inject primary antibody into third species of animal 5. kill animal and isolate secondary antibody 6. conjugate secondary antibody with flourophore 7. apply antibodies to brain of animal to be studied

Question 66

If you need an antibody to perform IHC for a chicken brain slice and you want to stain astrocytes on the slice, which antibodies won't work and why? A. Antibody against human GFAP B. Antibody against human NeuN C. Antibody against chicken GFAP

Answer 66

A - chicken has no human GFAP B - chicken has no human NeuN and NeuN doesn't stain astrocytes anyway

Question 67

If the primary antibody was made in a rabbit, which of the following secondary antibodies won't work for IHC staining? A. Donkey anti-rabbit Alexa-488 B. Goat anti-mouse Alexa-488

Answer 67

B