Tissue Prep And Staining Flashcards
What is the most common fixative and what is it good for?
Formalin and it’s good for viewing larger objects not fine cytological detail.
What are acid fixatives used to view?
Chromatin, nucleoli, and spindle fibers
Name 3 acid fixatives
Carnoy’s fluid
Zenker’s fluid
Bouins’s fluid
What are basic fixatives used for? And name 1 type
Mitochondria staining
Zirckle Erliki
What comes after fixing and what is it’s purpose?
Dehydrating, placing the tissue increasing strengths of ethanol. The purpose is to remove all water from the specimen because it will be embedded in paraffin (hydrophobic)
What shouldn’t you use ethanol for dehydration when your goal is to observe fats?
Because ethanol dissolves fats.
What is the purpose of clearing? And how do you accomplish clearing?
The goal is to remove the alcohol and you do so by replacing it with xylene or cedar oil.
What are the fixatives used for TEM?
Glutaraldehyde and osmium tetroxide
Describe the process of embedding
The specimen gets 3 paraffin baths
Placed in paraffin mold
Hardened by a cold water bath
Describe the embedding steps for TEM
Tissues are infiltrated with monomeric resin
Resin is polymerized
Tissue sample are less them 1mm cubed.
How do you prepare thin slices of specimen
Rotary microtome
How do you d sectioning for TEM
Sections are 50 to 150 nm
Sections so fragile must be floated onto plastic coated copper mesh grid
Holes in copper grid allow for beam of electrons to pass through tissue sample.
Diamond knives are used
How do you prep for staining?
Remove paraffin with xylene Remove xylene Stains are applied Dehydration Removal of alcohol with xylene Cement and cover slip
What is the H & E stain useful for?
Displaying structures, not chemical characteristics. H acts as a basic dye and stains light blue or purple and E acts as an acid dye and stains yellowish or pinkish color.
What are basic dyes
They bind with basic parts of the tissue (phosphate, sulfate and carboxyl groups).
What are tissues called that react with basic dyes?
Basophillic
What does basic dye binding depend on?
pH, and at a high pH all three basic groups are available (phosphate,sulfate carboxyl)
How do acid dyes bind with tissues?
Bye forming electrostatic bonds with cat ionic groups. Ex amino groups on proteins
Can different acid dyes be used in sequence to give different results? True or False
True
What is the name of any tissue that reacts with an acid dye?
Acidophillic
Give examples of acidic dyes
Acid fuchin
Aniline blue
Eosin
Orange G
What is metachromasia and give an example
When dye changes it’s normal color when binding with a tissue.
Ex: toluidine blue used to stain cartilage or mast cell granules
How do you stain tissue when using TEM? And give example
By using heaving metals that are electron dense. The electrons can be added during fixing, dehydration, or by soaking in ionic solutions after sectioning. Ex: Osmium tetroxide Uranyl nitrate Uranyl acetate and lead citrate For SEM platinum or gold may be used
What are histochemical techniques used for? Give 2 examples
Study the chemistry of cells and tissues.
Perls’ rxn: used to show presence of iron in tissues. Done by incubating tissue in potassium ferrocyanide + HCl. Insoluble blue precipitate of ferric ferrocyanide forms.
Ex: stains for lipids
Use frozen sections and use dyes that are soluble in lipids..Sudan 4, Sudan black, oil red O and Nile blue.
What are Schiff reagent reactions?
Reactions that depend on formation of aldehyde groups. They react with the aldehyde groups and form a deep pinkish color.
Give 2 examples of Schiff reagent reactions
Fuelgen reaction- hydrolysis with HCl exposes aldehyde group on deoxyribose
PAS- periodic acid cleaves bonds of carbs to form aldehyde.
Ex biopsies on patients with glycogenoses
What is best carmine?
Dye that shows glycogen deposits
What are 3 examples of stains?
Orecin and resorcin- reveal elastic materials
Silver impregnation- reticular fibers and basement membranes
Sudans- lipids
What is the purpose of fixing?
It preserves and hardens the specimen. All fixatives distort the specimen.
