Tissue Prep And Staining

Question 1

What is the most common fixative and what is it good for?

Answer 1

Formalin and it’s good for viewing larger objects not fine cytological detail.

Question 2

What are acid fixatives used to view?

Answer 2

Chromatin, nucleoli, and spindle fibers

Question 3

Name 3 acid fixatives

Answer 3

Carnoy’s fluid
Zenker’s fluid
Bouins’s fluid

Question 4

What are basic fixatives used for? And name 1 type

Answer 4

Mitochondria staining

Zirckle Erliki

Question 5

What comes after fixing and what is it’s purpose?

Answer 5

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)

Question 6

What shouldn’t you use ethanol for dehydration when your goal is to observe fats?

Answer 6

Because ethanol dissolves fats.

Question 7

What is the purpose of clearing? And how do you accomplish clearing?

Answer 7

The goal is to remove the alcohol and you do so by replacing it with xylene or cedar oil.

Question 8

What are the fixatives used for TEM?

Answer 8

Glutaraldehyde and osmium tetroxide

Question 9

Describe the process of embedding

Answer 9

The specimen gets 3 paraffin baths
Placed in paraffin mold
Hardened by a cold water bath

Question 10

Describe the embedding steps for TEM

Answer 10

Tissues are infiltrated with monomeric resin
Resin is polymerized
Tissue sample are less them 1mm cubed.

Question 11

How do you prepare thin slices of specimen

Answer 11

Rotary microtome

Question 12

How do you d sectioning for TEM

Answer 12

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

Question 13

How do you prep for staining?

Answer 13

Remove paraffin with xylene
Remove xylene
Stains are applied
Dehydration
Removal of alcohol with xylene
Cement and cover slip

Question 14

What is the H & E stain useful for?

Answer 14

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.

Question 15

What are basic dyes

Answer 15

They bind with basic parts of the tissue (phosphate, sulfate and carboxyl groups).

Question 16

What are tissues called that react with basic dyes?

Answer 16

Basophillic

Question 17

What does basic dye binding depend on?

Answer 17

pH, and at a high pH all three basic groups are available (phosphate,sulfate carboxyl)

Question 18

How do acid dyes bind with tissues?

Answer 18

Bye forming electrostatic bonds with cat ionic groups. Ex amino groups on proteins

Question 19

Can different acid dyes be used in sequence to give different results? True or False

Answer 19

True

Question 20

What is the name of any tissue that reacts with an acid dye?

Answer 20

Acidophillic

Question 21

Give examples of acidic dyes

Answer 21

Acid fuchin
Aniline blue
Eosin
Orange G

Question 22

What is metachromasia and give an example

Answer 22

When dye changes it’s normal color when binding with a tissue.
Ex: toluidine blue used to stain cartilage or mast cell granules

Question 23

How do you stain tissue when using TEM? And give example

Answer 23

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

Question 24

What are histochemical techniques used for? Give 2 examples

Answer 24

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.

Question 25

What are Schiff reagent reactions?

Answer 25

Reactions that depend on formation of aldehyde groups. They react with the aldehyde groups and form a deep pinkish color.

Question 26

Give 2 examples of Schiff reagent reactions

Answer 26

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

Question 27

What is best carmine?

Answer 27

Dye that shows glycogen deposits

Question 28

What are 3 examples of stains?

Answer 28

Orecin and resorcin- reveal elastic materials
Silver impregnation- reticular fibers and basement membranes
Sudans- lipids

Question 29

What is the purpose of fixing?

Answer 29

It preserves and hardens the specimen. All fixatives distort the specimen.