• study of microscopic anatomy
• Tissue is prepared, sectioned and stained, then visualized with a light or electron microscope

• for light microscopy, tissue is usually fixed with 4% formaldehyde in phosphate buffer. This permanenty cross-links proteins (by the formation of methylene bridges) so that the tissue does not degrade and the structure of the cells is stable
• we used rat brain tissue that was fixed by cardiac perfusion with 4% formaldehyde, and visualizing the tissue by light microscopy

1. rat deeply anesthetized w/ an overdose of sodium pentobarbital
2. thoracic cavity opened
3. perfusion needle placed through left ventricle & into aorta with right atrium cut
4. 100 ml ice cold saline perfused through the vasculature to remove the blood - liver & extremities should be pale
5. 400-500ml ice cold 4% formaldehyde in phosphate buffer is perfused through the vasculature; there will be tremors when the fixative begins to cross-link proteins
6. brain removed & post-fixed for 24-48 hours

• brains can be sectioned immediately with a vibrating microtome (25-50um) & mounted onto trated glass microscope slides (slides are slightly positive as brains are slightly negative). Can be stored free-floating in cryoprotectant at -20C until needed.
• Sections stored in the fridge in buffer
• alternatively brains can be embedded (like in paraffin) and stored at room temperature
• brain can also be placed in 20% sucrose/0.1M sodium phosphate buffer at 4C till it sinks. The brain is frozen in 2-methylbutane @ -30C.
• Too cold & brain splits, too warm and freezing is too blow and ice crystals form (swiss cheese brain)

• synthetic dye used to stain cell bodies in neuronal tissues purple, binds to acids as it is basic.
• used to identify tract of an electrode or cannula (placement, like for drugs)
• used to identify portion of brain destroyed by a neurotoxic lesion
• type of Nissl staining (Franz Nissl 1860-1919)
• Nissl substance/body = rough endoplasmic reticulum
• Nissl granules = ribosomes
• RNA = acidic and basophilic (base loving), so it binds basic dyes like cresyl violet
• glial cells are very small & if you still haeve blood, white blood cells will be labeled (not red blood cells or sperm though)

Myelin Staining

Golgi Stain

• Myelin Staining
• done with Luxol fast blue to visualize fiber tracts
• Golgi Stain
• developed by Camillo Golgi in 1873
• usses silver nitrate to stain whole neurons (but only stains some of them)
• used by Santiago Ramon y Cajal for neurondoctrine
• used to quantify # of dendritic spines under different conditions

• re-hydrate (in a descending series of alcohol baths)
• submerge in cresyl violet stain
• dehydrate (in an ascending series of alcohol baths)
• clear tissue with xylene
• 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

• 2 "sheets" of tissue - dentate gyrus and Cornu Ammonis (Ammon's horn) - folded onto each other
• neural circuitsstudied for LTP and LTD, processes critical in learning and memory
• major input from entorhinal cortexto dentate gyrus granule cells via perforant path
• axons of dentate gyrus = mossy fibers, synapse with CA3 pyramidal cells
• CA3 axons branch: leave hippocampus via fornix or form Schaffer collaterals which synapsewith pyramidal cells of CA1

• suprachiasmatic nucleus - biological clck - sleep/wake cycle
• supraoptic nucleus - water balance - big cells
• paraventricular nucleus - autonomic and neuroendocrine functions
• ventromedial nucleus - feeding - satiation (remove and get a fat rat)
• arcuate nucleus -  feeding, growth hormone regulation
• dorsomedial nucleus - feeding, drinking, circadian activity

Ventricles

Olfactory Bulbs

Caudate Putamen

Internal & external capsule

• Ventricles - produce & circulate CSF
• Olfactory Bulbs - sense of smell - well developed in a rat
• Caudate Putamen - movement - not separate in a rat (striatum)
• Internal & external capsule - fiber tracts

Nucleus Accumbens

Septum

Thalamus

Amygdala

Cerebellum & Pyramidal Tracts

• Nucleus Accumbens - reward
• Septum - "pleasure" center
• Thalamus - relay station for incoming sensory information en route to cortex
• Amygdala - fear learning and emotional behaviors
• Cerebellum & Pyramidal Tracts - motor control

• process of visualizing a specific protein within tissue using an antivody that binds selectively to that protein, and is conjugated to something that will enable it to be visualized
• requires use of different buffer solutions
• technique of visualizing an antigen (often protein) in or on cells within a tissue
• requires:
• tissue that has been processed appropriately
• monoclonal or polyclonal antibody to bind selectively to antigen of interest
• way  to visualize the antibody after binding

• keep the pH of a solution relatively stable, even when an acid or base is added
• made when a weak acid is mixed with its conjugate base
• H2CO3 carbonic acid and HCO3- bicarbonate acid is a classic buffer system that buffers blood pH
• Buffers work because there is an equilibrium between the acid (HA) and its conjugate base (A-)
• HA+H20 <=> H30+ + A-
• the factors that affect the pH are the Ka (equilibrium constant) of the reversible reaction, the relative concentrations of the acid and conjugate base, and temp
• at given temp, how likely is this equilibrim
• ph = -log10([H30+])

• phosphate buffer system used inside body cells
• dihydrogen phosphate ions (H2PO4-) are hydrogen ion donors and hydrogen phosphate ions (HPO4^2-) are hydrogen-ion acceptors
• H2PO4- <=> H+ + HPO4^2-
• pKa = 7.21, excellent for physiological systems
• internal cell pH = 6.9 to 7.4
• uffer most commly used for immunohistochemistry is a phosphate buffered saline, a phosphate based buffer with different salts at concentrations similar to those found in physiological systems

• first response to an invading pathogen - very quick and non-specialized, associated with inflammation
• can't keep up with dividing pathogen

• slow response the first time a pathogen is encounterd but faster for subsequent exposures
• very specific
• involves t-cells and b-cells (white blood cells/lymphocytes) that recognize specific antigens

• antibody generator
• anything that generates an adaptive immune response
• often proteins, but can be polysaccharides or lipids

• generated by plasma cells (differentiated B cells) of adaptive immune system
• glycoprotein that recognizes an dbinds to a speicfic antigen
• 5 classes
• IgG has highest concentration in serum & easy to get & generally used for immunohistochemistry
• IgG binds to an antigen (invading pathogen) and recruits other cells and molecules to destroy it
• each plasma cell secretes many copies of one specific antibody that recognizes one specific antigen

• IgG made of 2 heavy chains and 2 light chains
• Y shape w/ antigen binding site at end of each of 2 arms - called the Fab region (fragment, antigen binding)
• other end is not specific for binding antigen - Fc region (fragment, crystallizable)
• either whole IgG molecule is used or Fab fragments (made by digesting IgG with pepsin or papain that cleave polypeptide chains so there's just the binding site) used for immunohistochemistry

• antigen injected into an animal (often rabbit, also goats, horses, guinea pigs, hamsters, mice, rats, sheep, or chickens)
• animal generates different plasma cells tha tproduce different antibodies that recognize the antigen
• blood drawn from the animal to obtain antibody from serum

• comes from cells derived from a single B-cell line (clone)
• derived from mice
• mouse immunized with antigen and an antibody producing clone is isolated
• fusedwith a tumor cell to form a hybridoma that can be grown in culture & endlessly divide and produce antibody

• two ways to do it:
• antibody conjugated to an enzyme that catalyzes a reaction to form an insolublecolor product like peroxidase or alkaline phosphatase
• antibody is conjugated to a fluorophore that can be visualized more directly with fluorescent microscopy  (immunoflorescence)

• antibody conjugated to fluorophore that can be visualized by exposing it to light of a specific wavelength & detecting that light emitted by the fluorophore
• antibody can also bindto proteins and polysaccharides in tissue non-specifically. to help prevent this, we incubate in a solution that contains an excess of protein (bovine serum albumin) and polysaccharide (carrageenan)
• Triton X100 - detergent, will poke holes in membrane, making it more permeable to antibodies

• Sometimes primary antibody is not conjugated to an an enzyme or fluorophore so a secondary antibody is conjugated to fluorophor or enxyme to detect the primary antibody
• e.g. first antibody made in rabbit, second made in different animal and recogniz "rabbit" - goat anti rabbit
• secondary & sometimes tertiary antibodyes can also be used to amplify the signal

• relies on binding between avidin/streptavidin and biotin
• the secondary antibody is conjugated with biotin & pre-incubated with streptavidin in specific concentration ratio. complex added to the tissue and the streptavidin-enzyme complex binds to the biotynlated secondary antibody

• used to detect mRNA in cells
• based on DNA-RNA or RNA-RNA (stronger) hybridization of sense to antisense sequences
• "probes"canbe labeled with a fluorophore, radioactive, or antigenic tag
• radioactive labeling is useul when you want to compare the levels of mRNA in cells under different conditions. The sections can be exposed to x-ray film. More mRNA in cell = more radioactive probe hybridized = darker image, which can be quantified
• have reliable half-life
• fluorescent labeling is especially useful when you want to see if 2 mRNAs are colocalzed in same cell

• a very new method (2013) developed by Karl Deisseroth
• post-morten brian tissue cleared of lipids while proteins and nucleic acids remain
• uses clear hydrogel (acrylamide) scaffold/mesh to hold the remaining tissue components inplace when the lipid is removed
• lipid-free cells are transparent and are permeable to molecular markers like antibodies

• Identification
• Source
• manufacturer + catalog + lot # (vary from batch to batch, particularly polyclonal)
• research lab + antiserum code + bleed #
• Preparation
• what was the specific immunizing antigen? how much of it was put in (parts)?
• in which species was it raised?
• polyclonal/monoclonal?

Requirements for publishing in JCN

How has the specificity of the antibody been characterized?

• what doe sthe antibody stain on a gel?
• ideally, will stain one band of appropriate molecular weight - should be similar to protein's weight
• but often will have multiple bands, which is OK if the proten has multiple molecular configurations (GFAP)

Requirements for publishing in JCN

Immunostaining controls

• if molecule of interest isn't naturally in tissues, it's easy = show that there is no stain normally
• for other molecules, a knockout animal required & antibody/antiserum shouldn't stain tissue if  the molecule isn't there
• if knockout animal isn't possible -
• preincubate with excess of immunizing molecule
• show colocalization with the RNA that codes for the protein (in situ & immunohistochemistry)
• show similar staining patterns as a different antiserum/antibody raised against a differnt part of the molecule of interest
• show that the pattern of staining is identifical to previous description
• show that the staining is consisten with classic morphology annd distribution

Principles of Fluorescent Microscopy

Jablonski Diagram

• fluorescence is the result of a 3 stage process that occurs in fluorophors (also called fluors or florescent dyes)

Stage 1: excitation

• photon of energy is supplied by an extrenal source (mercury vapor lamp, metal hallide lamp, or a laser)
• energy absorbed by the fluorophore, creating an excited state (S1')
• process distinguished fluorescence from chemiluminescence, in which the excited state is produced by a chemical reaction - glow sticks

Stage 2: Excited-State Lifetime

• the excited state usually lasts for 1-10 nanosecs
• during this time, the fluorophore undergoes conformatonal changes and is subject to a number of different types of interactions with its molecular environment
• most important for fluorescent microscopy, the energy of S1' is partially dissipated, resulting in a lower energy exxcited state (S1) from which fluorescence emission originates

Stage 3: Fluorescence Emission

• the remaineder of the energy is emitted as a photon of energy, returning the fluorophore to tits grade state S0
• due to initial energy dissipation during the excited state lifetime, the energy of this photon is lower and therefore of longer wavelength than the excitation photon

• excitation and photon emission from a fluorophore is cyclical and it can be repeatedly excited until the fluorophore is irrevirsibly damaged
• once damaged, the flurophore no longer fluoresces - this is photobleaching
• photobleaching can be minimized by exposin gthe fluorophore to the lowest possible level of excitation light intensity for the shortest length of time. 

• excitation and emisson wavelengths are specific for each fluorophore
• distance between peak excitation and emission wavelengths is called the Stokes shift
• fluorophores with smaller Stokes shift give greater background signal because of the smaller differences between excitation and emission wavelengths

• DAPI
• binds to A-T rich region sof DNA, labels cell nuclei
• blue
• Alexa Fluor
• monoclonal
• green
• astrocytes
• Cy3 Conjugate
• polyclonal - rabbit
• orange 
• neurons