EXAM 3
Piriform cortex
A three layer cortex responsible for olfaction
CLARITY
A very new method (2013) developed by Karl Deisseroth (Stanford University School of Medicine)
Triton X-100
Allows antibody to pass through cell membranes to bind
20% sucrose/0.1 M sodium phosphate buffer 2-methylbutane @ -30ºC
Alternatively (this lab) brain can be placed in _____ at 4ºC till it sinks. The brain is frozen in ________ at ________.
differentiated B cell (plasma cell)
An antibody is produced by a what?
polyclonal antibodies
Antibodies that are secreted by different B cell lineages within the body
monoclonal antibodies
Antibodies that come from a single cell lineage
the protein (or part of the protein) is injected into an animal (rabbit) The animal will generate different plasma cells that produce different antibodies that recognize the different antigens on the protein Blood is drawn from the animal to obtain the polyclonal antibody (from serum).
Antibodies used in immunohistochemistry are referred to as polyclonal or monoclonal To generate a polyclonal antibody to a particular protein, what happens? (three things)
IgG
Antibody with the highest concentration in blood, and is the type generally used for immunohistochemistry.
An antigen (abbreviation of antibody generator)
Anything that generates an adaptive immune response. Are often proteins, but can be polysaccharides or lipids.
A monoclonal antibody
Comes from cells derived from a single B-cell/plasma cell line. Each plasma cell makes one type of antibody. Usually come from mice.
lower energy and longer wavelength
Compared with the light used to excite a fluorophore, the emission of a fluorophore has what kind of energy and wavelength?
tissue preparation Wash/re-hydrate sections in distilled water Submerge in cresyl violet stain Rinse in distilled water Dehydrate (in an ascending series of alcohol baths) Clear tissue with xylene (or Histoclear) Coverslip with Permount mounting medium After drying, observe sections using bright-field light microscopy Determine brain regions by comparison with pictures in a stereotaxic atlas
Cresyl Violet Staining - general method (nine things)
greenish yellow red region
Cy3 fluoresces what kind of color? Cy5 is fluorescent in the what region?
Liver is pale in color
During cardiac perfusion fixation, which of the following is/are a good sign that the tissue is being cleared of blood adequately?
fluorophore
During stage 2 of the Jablonski diagram, the what undergoes conformational changes and is subject to a number of different types of interactions with its molecular environment?
the brain will split
During tissue sectioning, if the brain is stored in too cold of temperature, what happens?
plasma cell
Each __________ secretes many copies of one specific antibody that recognizes one specific antigen
bregma (and the interaural line)
Each brain atlas schematic has measurements around the outside of the image. These show the dorsal-ventral and medial-lateral locations of areas on the section. The location of the section itself is described relative to what?
greater
Fluorophores with smaller Stokes shifts (right) give greater or smaller background signals?
see if 2 mRNAs are colocalized in the same cell
For In situ hybridization, fluorescent labeling is useful when you want to what?
compare the levels of mRNA in cells under different conditions.
For In situ hybridization, radioactive labeling is useful when you want to what?
fluorophore (e.g. FITC, Alexa Fluor) radioactive (e.g. 33P or 35S) antigenic (e.g. digoxigenin)
For In situ hybridization, the "probes" can be labeled with one of what three tags?
Mercury vapor lamp Metal halide lamp (our microscopes) Laser (eg for confocal microscopy)
For Stage 1 (Excitation) of the Jablonski diagram, what can be used as an external force used to supply a photon of energy? (three things)
This permanently cross-links proteins (by the formation of methylene bridges) so that the tissue does not degrade and the structure of the cells is stable
For light microscopy with histology, fixing the tissue in 4% formaldehyde in phosphate buffer does what?
4% formaldehyde in phosphate buffer
For light microscopy with histology, tissue is usually fixed with what in what buffer?
weighs more than usual for its age
Lesioning the ventromedial nucleus of the hypothalamus results in a rat that
Ventral posterior nucleus Ventral lateral nucleus Lateral geniculate nucleus Medial geniculate nucleus
List the four different subnuclei in the thalamus
•Suprachiasmatic nucleus (SCN) •Supraoptic nucleus (SON) •Paraventricular nucleus (PVN)
List the three different subnuclei in the hypothalamus
Stage 1: Excitation Stage 2: Excited-State Lifetime Stage 3: Fluorescence Emission
List the three stages of the Jablonski diagram
direct light to the camera
The light beam splitter can be pulled all the way out to do what?
in situ hybridization
The most suitable method to detect mRNA in the brain is what?
Golgi Stain (4)
Today, this stain is used to quantify the number of dendritic spines under different conditions (4)
true (6)
True of False? According to the protocol in the Lab Manual for "Immunohistochemistry 1" (Week 14, Lab 2), the antibodies would be used at a 1:8,000 dilution (6)
false (10)
True or False? After the brain sections have been incubated with the antibodies, they are heated to denature any unbound antibody to decrease background staining. (10)
false (6)
True or False? Carrageenan is a component of the blocking buffer for immunohistochemistry, and is there to prevent non-specific protein-protein interactions between the antibody and tissue. (6)
false
True or False? From Dr. Day's video showing the cresyl violet staining procedure, at the end of the staining procedure the sections were cleared in xylene.
true (3)
True or False? If you wanted to see the extent of a neurotoxic lesion of the brain, you could stain the tissue with cresyl violet. (3)
false (7)
True or False? Making a polyclonal antibody involves injecting an animal with different proteins so that more than one type of antibody will be made. (7)
true (9)
True or False? One way to measure pH of a solution is to determine the potential difference (voltage) between a reference system and the measuring system (9)
false (7)
True or False? The main buffering system inside cells is based on carbonic acid and its conjugate base, bicarbonate. (7)
false (you will not see it) (4)
True or false: You will see the processes of the neuron with cresyl violet staining? (4)
true(6)
True or false: the light beam splitter can be pushed in to direct all the light to the eyepieces? (6)
true (5)
True or false? The IgG class of antibody contains 2 regions that bind antigen (5)
Hippocampus
Two "sheets" of tissue folded on to each other. dentate gyrus DG and Cornu Ammonis (CA) or Ammon's horn.
Phosphate buffered saline Blocking buffer
Two buffers used in immunohistochemisty
In situ hybridization CLARITY
Two methods to visualize the brain in post-mortem tissue
Tissue is prepared, sectioned, and stained. Tissue is then visualized with a light or electron microscope
Two steps in histology
Direct Immunofluorescence (2)
Type of Immunofluorescence where the antibody is conjugated to a fluorophore that can be visualized by exposing it to light of a specific wavelength, and detecting the light that is emitted by the fluorophore (at a longer wavelength). (2)
PBS buffer plus
Type of blocking buffer (I think) Contains bovine serum albumin (BSA; a protein) and carrageenan (a polysaccharide) Contains Triton X-100 (a detergent)
zoonosis
Type of disease that is transferred from animals to humans.
to identify the tract of an electrode or cannula to identify the portion of the brain destroyed by a neurotoxic lesion
When would you use cresyl violet staining in research? (two things)
layer I (external) layer VI (internal)
Which layer is the most external layer? Which layer is the most internal layer?
the condenser condenses light on to a smaller area of the specimen
Which of the following is true for the condenser used in brightfield microscopy?
It is named for the person who developed it
Which of the following is true of Nissl staining?
luxol fast blue
Which of the following would you use to stain myelin, and reveal fiber tracts?
It is a landmark on the brain It is more rostral than lambda
Which two things are true for bregma?
The excitation and emission wavelengths
Which two wavelengths are specific for each fluorophore?
RNA is acidic and basophilic (base loving)
Why can RNA bind to basic dyes like cresyl violet?
because avidin/streptavidin can bind multiple biotin molecules
Why can the ABC method amplify the signal?
incubate in a solution that contains an excess of protein (bovine serum albumin) and polysaccharide (carrageenan)
With Direct Immunofluorescence, the antibody can also bind to proteins and polysaccharides in tissue non-specifically. To help prevent this, we do what?
different animal recognize "rabbit"
With immunohistochemistry, if you have a 1° antibody made in rabbit, your 2°would be made in a what and recognize what?
a secondary antibody that is conjugated to a fluorophore or enzyme
With immunohistochemistry, sometimes the primary antibody is not conjugated to an enzyme or fluorophore. In order to detect the primary antibody, what is used?
The Source: •manufacturer + catalog # + lot # •Research lab + antiserum code # + bleed #
What identification information about an antibody needs to be included in research? (technically 6 things)
"does the antibody/antiserum stain the tissue of interest from which the molecule of interest has been removed?"
What is the gold standard for immunohistochemistry?
a netwell
What is used to help drainage of fluid in immunohistochemistry?
Buffers
What keeps the pH of a solution relatively stable, even when an acid or base is added?
What was the specific immunizing antigen? In which species was it raised? Polyclonal or monoclonal? Also need to show that it stains at appropriate molecular weight on a protein gel (western blot)
What preparation information about an antibody needs to be included in research? (4 things)
A type III intermediate filament (IF) protein
What specific type of protein is GFAP?
Direct Immunofluorescence
What type of Immunofluorescence is used in this lab?
distinct
When multiple fluorophores are used in a single sample, it is important to choose those with spectra as what from each other as possible?
use the grippy strip to move a low power objective into position have a large working distance between the objective and the stage
When putting a slide on the microscope stage, you should do what two things?
in the left ventricle and into the ascending aorta
When rat brain tissue is fixed by cardiac perfusion, the perfusion needle should be placed where?
crystal violet
A stain for distinguishing Gram positive and Gram negative bacteria.
10 times as many
A solution with pH 1 has ___________________ hydrogen ions in it than a solution with pH 2.
DAPI (2)
4′,6-diamidino-2-phenylindole used in fluorescent microscopy (2)
antibody
A blood protein produced in response to and counteracting a specific antigen. Combines chemically with substances which the body recognizes as alien, such as bacteria, viruses, and foreign substances in the blood.
weak acid with its conjugate base
A buffer is made when you mix what together?
phosphate buffer system
A buffer system is used inside your body's cells Dihydrogen phosphate ions (H2PO4-) are hydrogen-ion donors (acid) and hydrogen phosphate ions (HPO42-) are hydrogen-ion acceptors (base) H2PO4- ⇌ H+ + HPO42-
Jablonski diagram
A diagram that shows electronic states and illustrates the process of fluorescence
DAPI
A fluorescent marker that binds to A-T rich regions of DNA, and hence labels cell nuclei
orange
A fluorophore that is excited with light in the green wavelength range would most likely emit light of which color?
Antibody
A glycoprotein that recognizes and binds to a specific antigen. Generated by plasma cells (differentiated B cells) of the adaptive immune system
amplifying host
A host organism (usually a vertebrate) in which a particular pathogen multiplies rapidly can serve to start or increase epidemics
reservoirs hosts
A host organism in which a particular pathogen resides but the host does not get sick
Western blot
A laboratory method used to detect specific protein molecules from among a mixture of proteins. This mixture can include all of the proteins associated with a particular tissue or cell type.
phosphate buffered saline
A phosphate based buffer that contains different salts at concentrations similar to those found in physiological systems used for washing tissue
GFAP protein (2)
A protein that is encoded by the GFAP gene in humans. (2)
GFAP protein
A protein that is expressed by numerous cell types of the central nervous system (CNS).
antigen
A protein that, when introduced in the blood, triggers the production of an antibody A molecule that is present at the outside of a pathogen, that can be bound to by an antigen-specific antibody
Adaptive immune response
A slow response the first time a pathogen is encountered (but faster for subsequent exposures). VERY specific. Involves T cells and B cells (types of white blood cells/lymphocytes) that recognize specific antigens
take pictures in black and white and pseudocolor them using the computer have the lights in the lab off/dim shutter the microscope light source when you are not viewing the slides
According to Dr. Day's video, in the lab, which of the following would we do when looking at slides with fluorescence microscopy? (three things)
AlexaFluor 488 and Cyanine 3 (CY3)
According to the PowerPoint presentation, which fluorophores are conjugated to the antibodies we use in the NRSC 2200 lab?
embedded room temperature
After rat brain dissection comes tissue sectioning: Alternatively brains can be _________, for example in paraffin, and stored at _________ until sectioning
vibrating microtome in the fridge in buffer
After rat brain dissection comes tissue sectioning: Brains can be sectioned immediately with a what? Where are the sections stored?
green
Alexa Fluor 488 fluoresces what color?
a fluorophore
Alexa Fluors; Dylight Fluors; fluorescein; cyanine dyes; rhodamine etc are examples of what?
mossy fibers
Axons of dentate gyrus (DG) are described as what?
CA3 pyramidal cells
Axons of dentate gyrus (DG) synapse with what?
equilibrium HA + H20 ⇌ H3O+ + A-
Buffers work because there is an ____________between the acid (HA) and its conjugate base (A-) in solution:
fornix Schaffer collaterals
CA3 axons branch: they either leave hippocampus via what? OR they form what which synapse with pyramidal cells of CA1
Ventromedial nucleus (VMH)
Caudal section of hypothalamus responsible for feeding
Arcuate nucleus (Arc)
Caudal section of hypothalamus responsible for feeding and growth hormone regulation
Dorsomedial nucleus (DMH)
Caudal section of hypothalamus responsible for feeding, drinking, and circadian activity
DNA blue
DAPI binds to _________ and fluoresces __________
Muscle tremors will occur After the process the body will be rigid
During cardiac perfusion fixation, which of the following are signs of good fixation? (two things)
The 2°antibody is conjugated with biotin. A reporter enzyme, conjugated with biotin, is pre-incubated with streptavidin in a specific concentration ratio. This complex is added to the tissue The streptavidin-enzyme complex binds to the biotinylated 2°antibody
Explain how the ABC method amplifies the signal (4 things)
Preincubate the antibody with an excess of the immunizing molecule Show colocalization with the mRNA that codes for the protein (in situ hybridization + immunohistochemistry) Show similar staining patterns as a different antiserum/antibody raised against a different part of the molecule of interest. Show that pattern of staining is identical to previous descriptions. (OK for classic markers) Show that the staining is consistent with classic morphology and distribution.
For molecules of interest that are naturally in tissue and with no knockout animal available, what is the next best thing to use to control for what you are staining? (five things)
using a knockout animal. The antibody/antiserum should not stain the tissue if the molecule it recognizes is not there.
For molecules of interest that are naturally in tissue, how do you control for what you are staining? (2 things)
phosphate buffer based mounting medium
For mounting tissue sections: Free floating tissue is placed in a _______ in a large petri dish.
true (2)
For mounting tissue sections: True or false: sections are dried (overnight) before being stained (2)
true
For mounting tissue sections: True or false: the slides are treated to give them a positive charge (eg with polylysine) so that the negatively charged tissue will stick to them?
a paint brush
For mounting tissue sections: What is used to mount the sections on to glass microscope slides?
molecular markers, like antibodies
For the CLARITY method, the lipid-free cells are transparent and are permeable to what?
tissue damage
For the Rat Brain Dissection: Scissors can be used to cut the skull as shown in the video, but this can lead to what?
4% formaldehyde
For the Rat Brain Dissection: The brain is then removed and post-fixed in what? (typically for 1-48 hours, depending on the procedure)
A Rongeur
For the Rat Brain Dissection: What can be used instead to peel back the skull and expose the brain and olfactory bulbs?
the head is cut off the skin cut the skull exposed
For the Rat Brain Dissection: When the fixative has been perfused, what three things happen so the brain can be removed?
cryostat or freezing microtome 35 μm
For tissue sectioning: The frozen brain is cut with a ________ into thin sections How big are our sections?
Free-floating
For tissue sectioning: What type of tissue sections can be stored in phosphate buffer at 4ºC or in cryoprotectant at -20ºC until needed?
the formation of methylene bridges
Formaldehyde permanently cross-links proteins by what?
Permount
From the video "Coverslipping brain sections" which of the following is used to adhere the coverslip to the slide?
Ventral posterior nucleus
Geniculate nucleus of the thalamus that has somatosensory relay and projects to POSTcentral gyrus.
Ventral lateral nucleus
Geniculate nucleus of the thalamus that is motor and projects to PREcentral gyrus
silver
Golgi staining relies on which metal ion?
Grams solute needed= Molarity x Liters x MW
Grams solute needed equation
1-hour
In immunohistochemistry, sections are incubated in blocking buffer for how long?
orbital shaker
In immunohistochemistry, sections are placed on an what to keep the tissue moving in the buffer?
Post-mortem brain tissue is cleared of lipids, while the proteins and nucleic acids remain
How does CLARITY work?
use a control
How does one ensure they are staining what they think they are staining?
5
How many classes of antibodies (also known as immunoglobulins) are there?
4: 2 heavy, 2 light
How many peptide chains is IgG made of?
green fluorescent protein (GFP) anatomical tracers bromodeoxyuridine (BrdU))
If the molecule of interest is not naturally in tissue, how do you control for what you are staining? (three things)
Fab region (fragment, antigen-binding)
IgG has a Y shape, and there is an identical antigen binding site at the end of each of the 2 arms. This end of IgG is called the what?
The Fc region (2)
IgG is a glycoprotein because which region is glycosylated? (2)
-tissue that has been processed appropriately -a monoclonal or polyclonal antibody to bind selectively to the antigen of interest -a way to visualize the antibody after binding
Immunohistochemistry requires three things
4% formaldehyde light microscopy
In NRSC 2200, we cresyl violet stain rat brain tissue that has been fixed by cardiac perfusion with what and visualize the tissue by what?
dichroic mirror
In a filter cube used for fluorescent microscopy, which part acts as a beam splitter, that reflects light of some wavelengths, while allowing other wavelengths through?
Blocking buffer
In immunohistochemistry used for "blocking" non-specific binding of antibody to tissue. i.e. helps to decrease background staining
Resolution refers to the ability to distinguish between 2 separate objects The magnification is a product of both the objective and ocular (eyepiece) lenses The objective lens inverts the image
In microscopy, which of the following is/are true? (three things)
7.4
In our lab, we use buffers with pH of what?
RNA (usually mRNA)
In situ hybridization is used to detect what in cells?
originates in the entorhinal cortex synapses with granule cells of the dentate gyrus
In terms of information flow in the hippocampus, the perforant path originates in the__________ and synthesizes with ___________.
avidin
In the ABC method of amplification in immunohistochemistry, a biotinylated secondary antibody binds to what?
acrylamide
In the CLARITY method of visualizing proteins and nucleic acids in the brain, the matrix which holds these components in place in the absence of lipids is what?
glass hook; paint brush
In the Mounting Floating Brain Sections video a ____________ was used to remove sections from the 12 well plate, and ____________ was used to mount the sections onto the slides?
a pH meter with glass electrode
In the lab, we measure pH with a what?
IgG (2)
In vivo, this antibody binds to an antigen on an invading pathogen. This triggers multiple processes that help eliminate the pathogen. (2)
perforant path
Information flow in hippocampus: Major input from entorhinal cortex to dentate gyrus granule cells via which path?
Cresyl violet
Is a synthetic dye used to stain the cell bodies in neuronal tissues purple
Immunohistochemistry (4)
Is the technique of visualizing an antigen (often a protein antigen) in or on cells within a tissue Is different from immunocytochemistry, which refers to cells isolated from tissue, e.g. cell culture (4)
Septum
Known as the "pleasure" center
Bregma and lambda
Landmarks on the skull, based on the fusion of the different skull bone plates. (two things)
learning and memory
Long term potentiation (LTP) and long term depression (LTD) are processes thought to be critical in what two things?
hippocampus (2)
Neural circuits in what brain region are studied a lot for long term potentiation (LTP) and long term depression (LTD)? (2)
rough endoplasmic reticulum ribosomes
Nissl substance/body = what? Nissl granules = what?
photobleaching
Once damaged, the fluorophore no longer fluoresces. This is called what?
blood
One classic physiological buffer system is carbonic acid (H2CO3) and its conjugate base bicarbonate (HCO3-). This is an important system that buffers the pH of what?
Golgi Stain (2)
Only stains some, not all, neurons. (2)
lowest possible level of excitation light intensity
Photobleaching can be minimized by exposing the fluorophore to the what level of excitation light intensity for the shortest length of time?
colocalization
Refers to observation of the spatial overlap between two (or more) different fluorescent labels, each having a separate emission wavelength, to see if the different "targets" are located in the same area of the cell or very near to one another.
Gold standard
Refers to the criteria by which scientific evidence is evaluated
Amygdala nuclei
Responsible for fear learning and emotional behaviors
Nucleus accumbens
Responsible for motivation, aversion, reward and reinforcement behaviors
Olfactory bulbs
Responsible for sense of smell Well developed in a rat Layers easy to distinguish with cresyl violet
Caudate putamen
Responsible for voluntary movement Not separate structures in a rat
pyramidal cells of CA1
Schaffer collaterals synapse with what?
A photon of energy (hνEX) is supplied by an external source. The energy is absorbed by the fluorophore, creating an excited state (S1')
Stage 1 (Excitation) of the Jablonski diagram involves what? (two things) •This process distinguishes fluorescence from chemiluminescence, in which the excited state is produced by a chemical reaction - eg glow sticks
1-10 nanoseconds
Stage 2: Excited-State Lifetime How long does the excited state in stage 2 of the Jablonski diagram usually exists for?
dissipated lower energy excited state
Stage 2: Excited-State Lifetime Most important for fluorescent microscopy, the energy of S1' is partially what? This results in a lower or higher energy excited state (S1) from which fluorescence emission originates?
lower longer
Stage 3: Fluorescence Emission for the Jablonski diagram Due to initial energy dissipation during the excited-state lifetime, the energy of this photon is _________, and therefore of __________ wavelength, than the excitation photon hνEX.
photon of energy hνEM
Stage 3: Fluorescence Emission for the Jablonski diagram The remainder of the energy is emitted as a___________, returning the fluorophore to its ground state S0.
Golgi Stain
Stain that was developed by Camillo Golgi in 1873. Uses silver nitrate to stain whole neurons, including processes.
Golgi Stain (3)
Stain used by Santiago Ramón y Cajal and inspired neuron doctrine (3)
Nissl staining - Cresyl Violet
Staining named for Franz Nissl (1860 - 1919).
Myelin staining
Staining used to visualize fiber tracts (e.g. Luxol fast blue)
4% formaldehyde in a sodium phosphate buffer tremors and body becomes rigid
Step five for cardiac perfusion involves perfusing with ice cold 400-500 ml of what? In this step, what happens when the fixative begins to cross-link proteins (20-30 min)?
clear the blood they should look pale
Step four for cardiac perfusions involves using a peristaltic pump to perfuse (via vasculature) with ice cold ~100 ml 0.9% saline and 0.1-1% sodium nitrite to do what? In this step, what should the liver and extremities look like? (about 5 min)
the left ventricle into the ascending aorta
Step one of surgery for perfusion fixation involves passing the perfusion needle through what?
iris scissors the right atrium
Step three of surgery for perfusion fixation involves using what make a large incision in what to allow blood and perfusate to drain?
to clamp the heart aorta
Step two of surgery for perfusion fixation involves securing the perfusion needle with one set of hemostats to do what? And a second set of a hemostats is used to clamp the what around the needle tip to prevent leakage?
Suprachiasmatic nucleus (SCN)
Subnuclei of the hypothalamus that is responsible for "biological clock"
Paraventricular nucleus (PVN)
Subnuclei of the hypothalamus that is responsible for autonomic and neuroendocrine functions
Supraoptic nucleus (SON)
Subnuclei of the hypothalamus that is responsible for water balance
stereotaxic surgery (2)
Targeting a specific brain region involves what? (2)
avidin/streptavidin and biotin
The ABC method is a very common method that relies on the binding between what?
disodium hydrogen phosphate
The blocking buffer contains which of the following?
Fc region (fragment, crystallizable).
The body of the Y shape of the IgG antibody other is not specific for binding antigen and is similar between different IgG molecules. What is this region called?
phosphate buffered saline (2)
The buffer most commonly used for immunohistochemistry is what? (2)
Stokes Shift
The distance between the peak excitation and emission wavelengths is called the what?
cyclical
The excitation and photon emission from a fluorophore is what? It can be repeatedly excited until the fluorophore is irreversibly damaged.
Ka (equilibrium constant) relative concentrations
The factors that affect the pH are the ________of the reversible reaction and the ________ of the acid and conjugate base
narrow wavelength range
The filter cube has a series of 3 filters, designed only to let light within what type of wavelength ranges through? Different filter cubes have different filters that are specific for a particular fluorophore.
Innate immune response
The first response to an invading pathogen. Very quick, and non-specialized. Associated with inflammation.
7.21
The pKa (pKa = -log10Ka) of the phosphate buffer system is what? It is an excellent buffer for physiological systems (internal cell pH = 6.9 to 7.4)
immunization
The process in which an individual's immune system becomes fortified against an agent.
Immunohistochemistry
The process of visualizing a specific protein within tissue using an antibody that binds selectively to that protein, and is conjugated to something that will enable it to be visualized
Fluorescence
The result of a three-stage process that occurs in fluorophores (also called fluors or fluorescent dyes).
positively; negatively
The sections stick to the glass slide because it is _________ charged, and the sections are ________ charged.
antisense sequence/strand
The strand that is the template during transcription, and eventually undergoes (typically, not always) translation into a protein.
Histology
The study of microscopic anatomy
there is no staining in a knock-out animal that does not express the protein that the antibody is meant to recognize
The very best method to show an antibody used in immunohistochemistry is labeling what you think, would be to show what?
Lateral geniculate nucleus (visual) Medial geniculate nucleus (auditory)
The visual thalamus The auditory thalamus
bovine serum albumin (BSA; a protein) AND carrageenan (a polysaccharide).
These bind weakly to the brain section and block non-specific binding of the glycoprotein antibody to the brain section
stereotaxic surgery
This form of surgery uses a 3D coordinate system, based on known landmarks (eg bregma) to locate the required area
In situ hybridization
This method is based on DNA-RNA or RNA-RNA hybridization of sense to antisense sequences
chemical
Tissue preparation for histology generally requires what fixation?
Determine the number of moles of solute present (1 mole = molecular weight of solute in grams) Determine the number of liters of solution present. Divide the number of moles of solute by the number of liters of solution
To calculate molarity (three steps)
The mouse is injected with the protein, which generates an immune response A specific antibody producing plasma cell is fused with a tumor cell to form a hybridoma. The hybridoma is grown in culture, and will endlessly divide and produce antibody.
To develop a monoclonal antibody against a protein, what happens? (three things)
filter cube
To facilitate distinction between fluorophores (whose spectra will still overlap to some extent), the light is passed through a what?
incubation
To keeps cells (or whatever you are testing in a lab) at a suitable temperature so that they develop
The sections can be exposed to x-ray film. More mRNA in cell = more radioactive probe hybridized = darker image, which can be quantified
Using radioactive labeling with in situ hybridization (two things)
enzyme fluorophore
Visualization of the Antibody by Light Microscopy: Antibody is conjugated to an _______ that catalyzes a reaction to form an insoluble color product OR Antibody is conjugated to a _________ that can be visualized more directly with fluorescent microscopy (immunofluorescence)
the smaller difference between excitation and emission wavelengths
What allows fluorophores with smaller Stokes shifts (right) to give greater background signals?
1. Make a lateral incision through the integument and abdominal wall. 2. Make an incision in the diaphragm and cut across the diaphragm exposing the heart. Make parallel cuts on either side of the ribs up to the collarbone. 3. Clamp the tip of the sternum with the hemostat and place the hemostat over the head.
What are the three steps once the thoracic cavity is opened during the cardiac perfusion process?
•The rat is first deeply anesthetized with an overdose of anesthetic (sodium pentobarbital) •The thoracic cavity is opened
What are the two steps mentioned for Tissue Preparation: Cardiac Perfusion?
astrocytes ependymal cells during development
What are two types of cells that express GFAP (including but not limited to)?
entorhinal cortex
What brings input to dentate gyrus of the hippocampus?
Secondary (and sometimes tertiary!) antibodies
What can also be used to amplify the signal?
a brain atlas
What do we use to help us determine how to locate a particular brain region if we are targeting it for an experiment. Eg to insert an electrode in a particular area?
Clear Lipid-exchanged, Acrylamide-hybridized Rigid, Imaging/immunostaining compatible Tissue hYdrogel
What does CLARITY stand for?
a clear hydrogel (e.g. acrylamide) scaffold/mesh
What does CLARITY use to hold the remaining tissue components in place when the lipid is removed?
Glial fibrillary acidic protein
What does GFAP stand for?
The Avidin-Biotin Complex
What does the ABC method stand for?
-Ideally, it will stain one band of appropriate molecular weight -But often will have multiple bands, which is OK if the protein has multiple molecular configurations like GFAP
What does the antibody stain on a gel in a Western blot test?
the brain was frozen too slowly
You have brain tissue that has been fixed by cardiac perfusion, and there are lots of little holes in the tissue, so that it looks a bit like swiss cheese. This is a sign that what happened?
Innate immune response Adaptive immune response
Your body has 2 types of immune responses
focus the image on the screen white balance the image so the computer knows what is white
after optimizing the image under the microscope, you should then optimize the image on the computer. To do this, you should do what two things?
pH = -log10([H3O+])
pH equation
sense sequence/stand
sequence of nucleotide chain that contains the information for protein synthesis the strand of DNA that has the same sequence as the mRNA
brightfield microscopy
the light beam splitter is usually used in a half-way position (50% light to camera and 50% light to eyepieces) for what?
Nissl staining (i.e. stains Nissl substance and Nissl granules)
•Cresyl violet staining is a type of staining?