NRSC 2200 FINAL WOOOOOOO

A fluorophore that is excited with light in the green wavelength range would most likely emit light of which color?

orange

An antibody is produced by a

differentiated B cell (plasma cell)

Compared with the light used to excite a fluorophore, the emission of a fluorophore has

lower energy + longer wavelength

What does DAPI bind to?

cell nuclei

What color does DAPI fluoresce?

blue

During cardiac perfusion fixation, how do you know fixation is working/going well?

Muscle tremors, body becomes rigid

During cardiac perfusion fixation, how do you know that the tissue is being cleared of blood adequately?

Liver is pale

Formaldehyde permanently cross-links proteins by

the formation of methylene bridges

What color does Alexa Fluor 488 fluoresce?

green

What color does Cy3 fluoresce?

orange

What does Alex Fluor 488 bind to/show?

neurons

What does Cy3 bind to/show?

astrocytes

What metal does Golgi staining rely on?

Silver

What does Golgi staining stain?

Whole neurons

What part of a filter cube acts as a beam splitter, that reflects light of some wavelengths, while allowing other wavelengths through?

Dichroic mirror

What does a dichroic mirror do?

beam splitter - reflects some wavelengths, allows other wavelengths to pass through

What's the purpose of the condenser used in brightfield microscopy?

condenses light on to a smaller area of the specimen

What's the perforant path of hippocampal information flow?

entorhinal cortex -> synapses with granule cells of the dentate gyrus

A biotinylated secondary antibody binds to

avidin

What's the matrix that holds proteins & nucleic acids in place in the absence of lipids after using the CLARITY method?

acrylamide

What happens to a rat if the ventromedial nucleus of the hypothalamus is lesioned?

It weighs more than usual for its age

ventromedial nucleus (VMH) - function

feeding

ventral posterior nucleus - function

somatosensory relay

Dorsomedial nucleus (DMH) - function

feeding, drinking, circadian activity

Parts of the hypothalamus

ventromedial nucleus, arcuate nucleus, dorsomedial nucleus, suprachiasmatic nucleus, supraoptic nucleus, paraventricular nucleus

Parts of the thalamus

Ventral posterior nucleus, ventral lateral nucleus, LGN, MGN

Caudate putamen - function

voluntary movement

arcuate nucleus (Arc) - function

feeding, growth, hormone regulation

amygdala nuclei - functions

fear learning, emotional behavior

nucleus accumbens - function

motivation, reversion, reward & reinforcement behzvior

ventral lateral nucleus - function

motor

What does the blocking buffer contain?

bovine serum albumin, Triton-X100, Carrageenan-lambda, 1X PBS

What does bovine serum albumin & carrageenan do in the blocking buffer?

Weakly bind to brain section, blocks non-specific binding of the glycoprotein antibody to the brain section

What does Triton X-100 do in the blocking buffer?

Allows antibodies to pass through cell membranes to mind

The distance between the peak excitation and emission wavelengths of a fluorophone is called....

the Stokes Shift

What allows a lens to magnify an image?

Refraction

What's the most suitable method to detect mRNA in the brain?

In situ hybridization

What's the BEST method to show that an antibody you used in immunohistochemistry is labeling what it's supposed to?

use a knock-out animal that doesn't express the protein that the antibody is meant to recognize - no staining should occur

What should you use to see the extent of a neurotoxin lesion in the brain?

cresyl violet

What should you use to identify the tract of an electrode or cannula?

cresyl violet

How do you make a polyclonal antibody?

inject protein into an animal, animal generates diff plasma cells that produce diff antibodies that recognize the diff antigens on the protein, draw blood from the animal to obtain it

What is a monoclonal antibody?

- cells derived from a single B-cell/plasma cell line. each plasma cell makes one type of antibody (usually derived from mice)

The IgG class of antibodies has how many regions that bind to antibodies?

2

How is a monoclonal antibody developed?

Mouse injected with protein, generates an immune response. The antibody-producing plasma cell fuses to a tumor cell, creating a hybridoma, which is then grown in culture to endlessly produce anitbody.

Where should the perfusion needle be placed during cardiac perfusion?

In the left ventricle, into the ascending aorta

Hypothalamic nucleus known as the "biological clock"

SCN

What are common enzymes that are conjugated to antibodies to enable visualization through producing an insoluble color product?

Alkaline phosphatase, peroxidase

What are common fluorophores that are conjugated to antibodies to visualize in fluorescent microscopy?

Alexa Fluors, Dylight Fluors, fluorescin, cyanine dyes, rohadmine

What do you use to stain myelin & reveal fiber tracts?

luxol fast blue

Why is it called Nissl staining?

Person who developed it - Franz Nissl

Brain tissue has been fixed by cardiac perfusion, but there's a lot of little holes in the tissue. What happened?

Brain was frozen too slowly

The energy that excites a fluorophone could be provided by...

mercury vapor lamp, metal halide lamp, laser

Secondary antibody

An antibody which specifically binds to a primary antibody, and is conjugated to a detectable marker

What is used in blocking buffer to prevent non-specific protein-protein interactions between antibody & tissue?

Bovine serum albumin

What does bovine serum albumin do in the blocking buffer?

Prevents non-specific protein-protein interactions

Name that fluorophores conjugated to antibodies that we used

AlexaFluor 488 & Cyanine 3

What component of the blocking buffer acts as the buffer?

disodium hydrogen phosphate

Why do brain sections stick to glass slides?

The slide is positively charged & the brain section is negatively charged

What's more rostral: bregma or lambda?

Bregma

After optimizing the image seen through the eyepiece of the microscope, how do you optimize the image on the computer?

Re-focus & white balance

What is the usual setting on the light beam splitter (control bar) for brightfield microscopy?

Half-way (50% light to camera, 50% light to eyepiece)

What objective do you use for brightfield microscopy?

2.5x

What layer of the cerebral cortex contains few neuronal bodies and looks pale with cresyl violet staining?

Layer I

What medium was used when putting CV sections on the slides?

phosphate buffer based mounting medium

2 ways to cut a fixed brain into sections

cryostat, freezing microtome

If a perfused brain splits after freezing, what happened?

too cold, frozen too fast

What should the screen on the microscope say?

TL_BF

What does Histoclear do?

De-fats cells, removes myelin

Is CY3 mono- or polyclonal?

monoclonal

Is AlexaFluor 488 mono- or polyclonal?

Polyclonal

What animal was the source of the CY3 antibody we used?

mouse

What animal was the source of the AlexaFluor 488 antibody we used?

rabbit

Septum - function

"pleasure" center

Piriform cortex - function

3 layer cortex; olfaction

Lateral geniculate nucleus - function

visual thalamus

Medial geniculate nucleus - function

auditory thalamus

Suprachiasmatic nucleus (SCN) - function

biological clock

supraoptic nucleus (SON) - function

water balance

paraventricular nucleus (PVN) - function

autonomic and neuroendocrine

What was used to fix the rat brain tissue?

4% formaldehyde

What does Nissl staining stain?

rough ER & ribosomes

What does cresyl violet stain?

RNA

Why does cresyl violet stain RNA?

RNA is acidic and loves bases like CV

What are the two types of immune response?

Innate & adaptive

What is the first response to an invading pathogen that's quick, non-specialized, & associated with inflammation

Innate immune response

What is the slow response seen when the first time a pathogen is encountered, and becomes faster through repeat exposures?

Adaptive immune response

adaptive immune response vs innate immune response

adaptive = slower, very specific, T cells, B cells

innate = quick, non-specialized

What is the term for "anything that generates an adaptive immune response"

antigen

Antigens can be

proteins, polysaccharides, lipids

What is the term for a "glycoprotein that recognizes and binds to a specific antigen"?

Antibody

What shape is IgG?

Y

What are the specific antigen-binding sites of IgG called?

Fab region (fragment, antigen-binding)

What are the sites of IgG called that don't bind antigen, and is generalizable throughout IgG molecules?

Fc region (fragment, crystallizable)

What's the purpose of secondary antibodies?

Detect primary antibodies and/or amplify the signals

If a knockout animal isn't available to test the antibody, what are the next best methods?

Preincubate antibody w an excess of immunizing molecule; show colocalization w mRNA that codes for the protein; show similar staining patterns to a similar antibody; show that stain pattern is identical to other descriptions; show that staining is consistent w classic/known morphology/distribution

What does in situ hybridization use?

- DNA-RNA or RNA-RNA hybridization of sense to antisense sequences

What's labelled w a fluorophore or antigenic tag in in-situ hybridization?

probes

What would you use to compare LEVELS of mRNA in cells under diff conditions?

Radioactive labelling

What would you use to see if 2 diff mRNAs are found in the same cell?

Fluorescent labelling