Specialized preparation techniques and labeling
 
Why have specialized techniques?
Labels:
Need to know the composition of the structure/organelle and or phase of cycle.
Identification of a questionable structure (e.g. microsomes, peroxisomes, vesicles)
Follow cytochemical pathway through a time course.
Localize genetic products, introduced compounds.

Cryo-fixation:
Unable to obtain adequate preservation of structures by conventional methods
- structures are ephemeral (membrane fusion) or events occur quickly.
- the structures are fixative sensitive (e.g. vesicles, enzymes, water soluble components)
- Structural organization needs to be viewed by TEM vs SEM because of resolution.
- Removal of water changes the topography/morphology of the structures (e.g. clays or soils, foods such as pnut butter, cheese, creams etc…)


Prep Techniques
Shadowing and Replicas –
Direct: TEM sample is coated and viewed
Indirect: Replica is made and viewed. Can be two step or one step method.
For SEM: Vinyl or plastic replica can be made by infusion into tissue, then original tissue is removed (negative cast)
SEM: A replica of the surface is made, then prepared for viewing.

Low temperature techniques
Freeze Fracture – Sample is rapidly frozen, fractured and a replica is produced. The sample is typically etched away to leave the replica. Viewed with TEM
Cryo-fixation – Can use a variety of methods to fix the tissue prior to substitution (removal of water). Plunge and jet are the most common. Spray, HPF, slam are not as common. Each has variable success for differing tissues and sizes. SEM and TEM.
Cryo-ultramicrotomy – Used in conjunction with cryo-fixation to maintain frozen state of sample. The sections are then kept frozen for viewing or processed to dryness for conventional viewing.
Freeze drying – Easiest and most common technique for SEM. Sample is quick frozen and then placed under high vacuum while kept frozen. Water is slowly removed until completely dry.

Labeling
Autoradiography – Labeling with an isotope, then doing a reaction which deposits metal at the radioactive sites. Good for following an introduced substance through a cycle. Pulse/chase experiments.
Typical probes include tritated components, P32, etc… The label is added pre-fix and visualization occurs post embedding. Viewed with TEM
Lectin labeling – lectins are small molecular weight components that have variable affinities for sugars.
Important to have controls. Typically added post-embed. Typically TEM, possible to use with SEM
Cytochemical labeling to identify carbohydrates, enzymes, proteins, etc… Chemical reactions require the precipitation of heavy metal for visualization. Can be pre-embedding or (more commonly) post-embedding. Typically TEM
Immunolabeling – Use of either polyclonal or monoclonal antibodies to label components. Visualization requires colloidal gold or HP conjugation to the antibody or a secondary antibody. TEM or SEM
Silver Enhancement – can be added to any label listed above to increase size of label. Also allows correlative studies at the light level. TEM