Staining

Image formation

1. wavelength contrast--> differences in color 

2. phase contrast--> difference in wave alignment 

3. amplitude contrasts--> differences in intensity

  see #2,3 in TEM scope,  (see colors only in light scope)

3 factors in amplitude contrast

1. absorption -->requires # of collisions the heavier the atom the more absorbent

2. diffraction--> reason for fringes around objects--use for accentuating contrast

3. scattering -->neg charged e interacts with sample
 

elastic scattering by nucleus--
        large deflection of path but no loss of energy ==> most lost in column, unseen in image
inelastic scattering by electron collisions -->e orbital collision, both e loose energy and change direction
        deflection of path, loss of energy ==> see in image

use negative stain objects light against dark background
 
for small particulates, don't need to fix and embed

positive staining increased density against light background

staining defined by 1) number of sites per molecule and 2) density of sites in section=>thickness of section

Contrast is a function of section thickness and density of stain
Thinner sections have less overlapping material and better resolution but poor contrast due to too little stain present

Thicker sections have less contrast and decreased resolution because too much stain is present everywhere. There is also more cell material obscuring details.

To increase contrast in the TEM, use a smaller aperture to reduce inelastic scattering contribution to image formation

 use heavy metals==>more scattering with increased atomic weight will vary with tissue, preparation, plastics etc

plant tissue stains differently than animal--needs less time

 glut fixed lipids extracted in alcohols so see poor or no lipid staining

osmium lipids are darkly stained

fixatives can also act as stains-- osmium and uranyl acetate for lipids

some buffers will ppt stains, such as phosphate and UA

pH heavy metal stains form polynuclear complexes at increasing pH and can then ppt as hydroxide for more contrast

interactions can be either ionic or electrostatic

stain penetration

pre embed vs post embed----either in fixation or in post embedding some stains only work at surface of cells, such as rhuthenium red for mucopolysac.

Uranyl Acetate will stain throughout tissue

 pre embed vs post embed-- it is best to stain after fixation
    1. reduce extraction of tissue by stain
    2. more uniform staining
    3. less alteration of structures by stain
    4. more easily controlled

stain specificity will vary with concentration, pH, times

both common stains, UA and lead are saturation stains with little differentiation

use uranyl acetate at acid pH

lead at alkaline pH

Cytochem

fix-. maintain 3D protein configuration & stabilize cellular organization

may change shape

may cross link sites

may inhibit access by other cross links


Aldehydes

formaldehyde links are weaker than glutaraldehyde, so preferred

make membranes leaky

aids access and penetration

con--allows degradation/digestion by proteolysis

Osmium

strong oxidant

heavy metal

stain


Buffers

may ppt reaction product-->phosphate will precipitate lead

may interfere with charges on groups of interest-->phosphate alters negatively charged molecules  

Markers
may not be easily resolvable

anything large enough to see can't move inside of cell

to improve access, cut slices of tissue

Some cytochemical examples:
Osmium at elevated temp and time ==>produce a precipitate in golgi

silver plus thiocarbohydrazide ==> carbohydrates stained black

phosphatases localized in different endomembrane compartments

Typical cytochemcal reaction, with controls:
enzyme (in tissue block)+substrate +buffer + marker(trapping agent)= product

enzyme (in tissue block)+poison +substrate +buffer + marker(trapping agent)=nothing (enzyme actiion has been poisoned)

enzyme(in tissue block)+buffer + marker(trapping agent)= nothing (without substrate present)

acid phosphatase enzyme in tissue+B-glycerophosphate+buffer at pH 6+ lead nitrate==> release of P04 produces lead phosphate ppt

change pH to pH8, see alkaline phosphatase activity

glucose -6-phosphatase, using glucose-6-phosphate substrate-->ER

inosine diphosphatase--> trans golgi

acid phosphatase--> lysosomes

need to do time points to ascertain best staining with minimum background

Antibody localization

Antibody is a protein that will specifically bind to a specific protein or amino acid sequence

Make antibodies by exposing animal to a foreign protein of interest (antigen, e.g.--microtubule or muscle protein)

Animal responds by making antibodies that will target the foreign material.

when antibody binds to foreign material, triggers a reaction that removes foreign particles from the body
How can you visualize--attach a marker Colored or fluorescent for light microscope; e dense particles for TEMDirect vs Indirect

Direct: single step antibody with marker attached binds to a single antigen

Indirect: two step process

1. expose sample to primary antibodies in soln(no marker attached)
rinse sample with buffer

2. expose sample with primary attached to antigens to secondary antibodies with markers attached

2ndary will attach to multiple places on primary, so multiple markers end up attached to a single antigenPrimary vs secondary antibody

Primary

1. isolate protein of interest

2. inject into animal

3. after several weeks, collect blood

4. isolate and identify antibodies that bind to your protein of interest

Secondary 1. isolate antibodies from same kind of animal used to produce primary

2. inject into a different kind of animal (unrelated genus)

3. after several weeks, collect blood

4. isolate and identify antibodies that bind to your antibodies of the primary animal

5. attach a fluorescent (LM) or e-dense (TEM) label to these secondary antibodies