Unit 3 Flashcards
Histology
Study of microscopic anatomy
- tissue is prepared, sectioned and stained, then visualized with a microscope
Tissue preparation
-Usually requires chemical fixation
- Light microscopy - 4% formaldehyde in phsophate buffer
What does the 4% formaldehyde do?
Permanently cross-links proteins by formation of methylene bridges so that the tissue does not degrade
Perfusion fixation
Removes blood from an animal
What is first done to the rat before perfusion fixation?
The rat is first deeply anesthetized with an overdose
How is the thoracic cavity opened?
- Make a lateral incision through the integument and abdominal wall
- 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
- Clamp the tip of the sternum with the hemostat and place the hemostat over the head
What is done after the thoracic cavity opening?
- A perfusion needle is passed through the LEFT ventricle into the ASCENDING aorta
- The needle is secured with one set of hemostats to clamp the heart. A second set of hemostats can also be clamped around the aorta to prevent leakage.
- Iris scissors are used to make a large incision in the RIGHT atrium to allow blood and perfusate to drain
After inserting the perfusion needle…
- A periplastic pump perfuses ice cold 0.9% saline and 0.1-1% sodium nitrate to clear the blood (LIVER AND EXTREMITIES SHOULD BE PALE) - 5 min
- Then perfuse with 4% formaldehyde in a sodium phosphate buffer. You will see tremors when the fixative begins to cross-link proteins and the body will become rigid (20-30 min)
What signs show that the perfusion was done correctly?
Pale liver and extremities; rigid body and tremors
What happens after perfusion of blood?
Head is cut off, skin is cut and skill is exposed so that the brain can be removed
What is usually used instead of scissors to cut the skull?
Rongeur – peels back skull and exposes brain and olfactory bulbs
Where is brain placed after removal?
4% formaldehyde (1-48 hours after procedure)
What are the three methods of sectioning tissue?
- Immediately with a vibrating microtome and sections are stored in a fridge with a buffer
- Brains can be embedded in paraffin and stored at room temperature until sectioning
- Brains can be placed in 20% sucrose/0.1 M sodium phosphate buffer at 4C until it sinks. Brain is frozen in 2-methybutane at -30C
Why is the temperature important for freezing and storing the brain?
Too cold and the brain will split; too warm/frozen too slowly and the freezing is too slow so ice crystals form - the brain will look like swiss cheese!
How thin were the sections for cresyl violet staining?
35 micrometers
How can free-floating tissue sections be stored?
In phosphate buffer at 4C or in cryoprotectant at -20C until needed
Steps to Mount Tissue Sections
- Free floating tissue is placed in a phosphate buffer based mounting medium (PBS - PB saline) in a large petri dish
- A paint brush is used to mount the sections on to glass microscope slides
- Slides are treated to give them a positive charge (with polylysine) so that negatively charged tissue will stick to them
- Sections are dried overnight before being stained
What can you use to cut tissue sections?
- Cryostat (what we used)
- Freezing microtome
Why are tissues negatively charged?
Proteins are negatively charged!
Immunohistochemistry
Process of visualizing a specific protein within tissue using an antibody that binds selectively to that protein and is conjugated to something that will allow it to be visualized
What is required for immunohistochemical method?
Buffers - they keep the pH of a solution stable no matter what
What is pH measured on?
Log scale: a difference of 1 pH unit is a 10x difference in H+ ions
What are buffers made of?
Weak acid and its conjugate base
What is the classical buffer system?
Carbonic acid and bicarbonate - buffers blood pH
Where are phosphate buffers used?
Inside cells
What is the phosphate buffer made of?
Dihydrogen phosphate ions (acid) and hydrogen phosphate ions (base)
Most commonly used buffer in immunohistochemistry
Phosphate buffered saline (PBS)
What does a blocking buffer contain?
- PBS
- Bovine serum albumin (BSA) and carrageenan (bind weakly to the brain and block non-specific binding of the antibody to other proteins)
- Triton X-100 (detergent - allows antibody to pass through cell membrane to bind)
What pH of buffers do we use in the lab?
7.4
Molarity calculation
moles of solute / liters of solution
Grams of solute =
Molarity x liters x MW
% solutions important notes
- w/v or v/v
- 0.9% has 0.9 grams/mL in 100 mL
Two types of immune responses
Innate immune response and adaptive immune response
Innate immune response
First response to invading pathogen, very quick and non-specialized
Adaptive immune response
Slow response the first time a pathogen is encountered, but becomes but faster with subsequent exposure; very specific, involves B and T cells that recognize specific antigens
Antigens
Anything that generates an adaptive immune response
Antibody
glycoprotein that binds to a specific antigen generated by differentiated B cells
Immunoglobulin
5 classes of antibodies
IgG
Immunoglobulin that is the highest concentration in blood; usually most commonly used in immunohistochemistry
What does IgG do in vivo?
Binds to an antigen on an invading pathogen
What letters does an antibody resemble?
Y
How many peptide chains are there in an antibody?
4: 2 heavy and 2 light
Where are the antigen binding sites?
On the ends of the Fab region (Fragment, antigen binding)
Where on the antibody is not specific to an antigen?
Fc (fragment, crystallized)
Why is an antibody a glycoprotein?
The Fc region is glycosylated
Two types of antibodies used in immunohistochemistry
Monoclonal and Polyclonal
Polyclonal antibodies
Many types of antibodies against one protein
Monoclonal antibodies
One type of antibody from a single type of B-cell/plasma cell
How to make polyclonal antibodies
Protein is injected in an animal (often a rabbit). The animal will generate different plasma cells that recognize the different antigens on the protein. Blood is drawn and you get the polyclonal antibody
How to make monoclonal antibodies
Mouse/animal is injected with protein, then a specific antibody producing plasma cell is fused with a tumor cell to form a hybridoma, which endlessly divides in culture. Antibodies are then isolated
What does immunohistochemistry require?
- Tissue that was appropriately processed
- Monoclonal/polyclonal antibody
- A way to visualize the antibody
What are ways to visualize antibodies?
Use a light microscope and conjugate the antibody with either an ENZYME (catalyzes a reaction to form an insoluble color like peroxidase or alkaline phosphatase) or a FLUOROPHORE (visualized more directly - Alexa fluors, Dylight flours, cyanine dyes)
Direct immunofluorescence
Antibody is directly conjugated to a fluorophore that can be visualized when exposed to light of a specific wavelength
What is a drawback of direct immunofluorescence and what can you use to not have that happen?
Antibody can bind to protein and polysaccharides non-specifically. Therefore, we add BSA (excess protein) and carrageenan (excess polysaccharide) into solution so that there is blocks on non-specific proteins
Fluorophores that we used
Alexa Fluor #488 (neurons) and CY3 (astrocytes)
Name of antibodies that we used
Proprietary pan neuronal marker (polyclonal; neuron) and GFAP (monoclonal; astrocytes and glia in cerebellum)
Indirect immunohistochemistry
A secondary antibody is conjugated to a fluorophore or an enzyme that detects the primary antibody, which allows for large scale amplification of a signal
ABC Method of Immunohistochemistry
Avidin-Biotin Complex; avidin/streptavidin binds to biotinylated secondary antibody which then give signal with a reporter enzyme
ABC Method Description
Secondary antibody is conjugated with biotin. A reporter enzyme, conjugated with biotin, is preincubated in streptavidin in a specific concentration ratio. This complex is added to the tissue and the streptavidin-enzyme complex binds to the biotinylated secondary antibody
How do we know if the antibody is staining what it should be?
- JCN gold standard: check in a tissue that doesn’t have that molecule OR use a knockout animal - the antibody should NOT stain the tissue if the molecule it recognizes is not there
- If no knockout animal, use controls
Complete information needed about the antibody
Identification (source) and preparation (what was the specific antigen, in which species was it raised, polyclonal or monoclonal) and it needs to show that is stains at appropriate molecular weight on a protein gel (western blot)
What does the antibody stain on a gel?
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!)
Controls other than a knockout mouse for an antibody
- Preincubate the antibody with excess of immunizing molecule (Abs should bind here, but then not bind in actual tissue)
- Show colocalization with the mRNA that codes for the protein (would be hard because mRNA is in nucleus but protein could be far away)
- Show similar staining patterns as a different antibody raised against a different part of molecule of interest
- Show that pattern of staining is identical to previous descriptions (OK for classic markers)
- Show that staining is consistent with classic morphology and distribution
What are other methods of visualizing the brain in post-mortem tissue?
- In situ hybridization
- CLARITY
What does in situ hybridization detect?
RNA, using DNA-RNA/RNA-RNA hybridization
How are these strands labelled?
Probes are labeled with fluorophore, antigenic, or radioactive tags
Radioactive labelling in in situ hybridization
Useful to compare relative levels of mRNA (more mRNA = more radioactive probes hybridized = darker image)
Fluorescent labelling in in situ hybridization
Useful to see if 2 mRNAs are colocalized in the same cell
CLARITY
Removal of lipids while nucleic acids and proteins remain
CLARITY - how?
A clear hydrogel scaffold holds remaining tissue in place when lipid is remove; the lipid free cells are transparent and permeable to molecular markers like antibodies
The process of fluorescence
Three stage process that occurs in fluorophores
Jablonski diagram (definition)
Shows electronic states of fluorescence
Jablonski (step 1)
Excitation: photon of energy is supplied by an external source (microscopes) and is absorbed by the fluorophore, creating an excited state (S1’)
What is chemiluminescence?
Excited state is produced by a reaction rather than photons (i.e. glow sticks)
Jablonski Diagram (step 2)
Excited-State Lifetime: excited state (S1’) exists for 1 to 10 nanoseconds, during which the fluorophore undergoes conformational changes and goes from an energy source of S1’ to S1 (lower energy excited state)
Jablonski Diagram (step 3)
Fluorescence emission: remainder of the energy is emitted as a photo of energy returning the fluorophore it its ground state. Due to initial energy dissipation during step 2, the energy of this photon is lower, and therefore of longer wavelength that the excitation photon
Photobleaching
Irreversible damage to fluorophore = can’t fluoresce anymore!
How to minimize photobleaching
Expose fluorophore to the lowest possible level of excitation light intensity for the shortest length of time (tin foil!)
Stokes shift
Distance between the peak excitation and emission wavelengths
True or false: excitation and emission wavelengths are specific for each fluorophore
TRUE
Is it better to have a larger or smaller stokes shift?
Larger - less background signal
Can overlap exist between fluorophores?
Yes, a lot of overlap can exist when using multiple fluorophores
What can you do to help differentiate between multiple fluorophores?
- Choose fluorophores with spectra as distinct as possible
- Use a filter cube (series of 3 filters, allowing only light within a narrow wavelength range to go through)
Parts of a filter cube
- Excitation filter (high energy shorter wavelength)
- Dichroic mirror (reflects excitation, hits object, but does not reflect emission because it is lower energy)
- Emission filter (narrows range even further of the emitted light)
Excitation light is ____ energy and _____ wavelength
High; shorter
Emission light is _____ energy and _____ wavelength
Low; longer
Cresyl violet staining
synthetic dye used to stain the cell bodies in nervous system purple
Cresyl violet is a type of ____ staining
Nissl - nissl bodies = RER, nissl granules = ribosome
What does cresyl violet bind to and why?
Binds to RNA because it is acidic and basophilic while cresyl violet is a basic dye!
When does one use cresyl violet?
In research - identify tracts of electrodes or identifying the portion of the brain destroyed by a neurotoxic lesion
Luxol Fast Blue
Myelin staining; used to visualize fiber tracts
Golgi Stain
Developed by Camillo Golgi to use silver nitrate to stain whole neurons (stains whole, but not all neurons); used today to quantify the number of dendritic processes under different conditions
General method of cresyl violet staining
- Prepare tissue
- Wash/re-hydrate sections in distilled water
- Submerge in cresyl violet stain
- Dehydrate (in ascending alcohol baths)
- Clear tissue with xylenes or histoclear
- Coverslip with permount mounting medium
- After drying, observe section using bright field microscopy and use a rat atlas to determine areas of the brain
Bregma and Lambda
A landmark on the skull based on the fusion of different skull bones
When does one use rat atlas?
To determine how to locate a particular brain region - i.e. in stereotaxic surgery
True or false: Bregma is a point on the brain
FALSE - it is a point on the skull
How many layers are in the cerebral cortex?
6; 1 is outermost and 6 is innermost
Hippocampus structure
Two sheets of tissue, a dendate gyrus and CA areas that are folded on one another
What processes are thought to be important in the hippocampus?
LTD and LTP - processes crucial in learning and memory
Information flow in the hippocampus
- Entorhinal cortex to DG granule cells (via perforant path)
- DG to CA3 pyramidal cells (via mossy fibers)
- CA3 axons branch and either leave hippocampus via fornix OR synapse with CA1 pyramidal cells (via Schaffer collaterals)
Thalamus Nuclei
- Ventral posterior
- Ventrolateral
- Lateral Geniculate
- Medial Geniculate
Function of ventral posterior nucleus
Somatosensory relay (to post central gyrus)
Function of ventral lateral nucleus
Motor relay (to precentral gyrus)
Function of lateral geniculate nucleus
Visual thalamus
Function of medial geniculate nucleus
Auditory thalamus
Hypothalamus nuclei
- Suprachiasmatic (biological clock)
- Supraoptic (water balance)
- Paraventricular nucleus (autonomic and neuroendocrine functions)
- Ventromedial (feeding)
- Arcuate (feeding, growth hormone regulation)
- Dorsomedial nucleus (feeding, drinking, circadian rhythm)
Function of olfactory bulbs
Sense of smell - very well developed in a rat
Caudate putamen
Voluntary movement - NOT SEPARATE STRUCTURES IN A RAT
Nucleus accumbens
Motivation, aversion, reward, and reinforcement mechanisms
Septum
Pleasure center
Amygdala nuclei
Fear learning and emotional behaviors
Piriform cortex
3 layer cortex - olfaction
