Tissue prep and staining

Question 1

what are the steps needed to prepare tissues for observation

Answer 1

fixing, dehydration, removal of alcohol, embedding

Question 2

what does fixation prevent

Answer 2

further deterioration of the specimen and helps to harden the tissue prior to embedding and sectioning

Question 3

what is the main downfall of fixation

Answer 3

it radically distorts the specimen

Question 4

what is one of the most widely used fixing agents

Answer 4

Formalin

Question 5

what does formalin do

Answer 5

fixing agent that reacts with the amino acids of the tissue proteins and stabilizes tissue structure to prevent further deterioration

Question 6

what do acid fixatives fix? don’t fix?

Answer 6

fix: chromatin, nuclei and spindle fibers

don’t fix: mitochondria and nucleoplasm

Question 7

name three acid fixatives

Answer 7

carnoy’s fluid
zenker’s fluid
Bouin’s fluid

Question 8

what is Carnoy’s fluid good for

Answer 8

good general fixative and is useful for preserving glycogen in animal tissues

Question 9

what is Zenker’s fluid good for

Answer 9

useful when sharp histological detail is desired, but must be washed out carefully to prevent the precipitation of black crystals

Question 10

what is Bouin’s fluid good for

Answer 10

widely used general fixative that gives cytological detail

Question 11

when are basic fixatives usually used

Answer 11

when mitochondrial staining is desired

Question 12

what is the main downfall of basic fixatives

Answer 12

chromatin is dissolved

Question 13

what are 2 main fixatives for TEM

Answer 13

Glutaraldehyde: preserves proteins by cross-linking them

Osmium tetroxide: reacts with lipids and imparts electron density to cell and tissue structure

Question 14

why must dehydration occur for tissue fixing and embedding?

Answer 14

because the tissue sample will eventually be embedded and infiltrated with a hydrophobic material (usually paraffin)

Question 15

what is clearing?

Answer 15

consists of replacing the alcohol with an agent such as xylene or cedar oil

Question 16

what are the steps of embedding

Answer 16

1. the tissue specimen is moved sequentially through several melted paraffin baths
2. after the final bath the specimen is placed in a mold that is then filled with melted paraffin
3. the paraffin mold is rapidly hardened by placing it in a cold water bath

Question 17

steps to prepare for staining

Answer 17

1. paraffin must be removed from the section, which is now mounted on a microscope slide (accomplished with xylene)
2. xylene must be removed using a graded series of alcohol down to water
3. stains are then applied and the section is again dehydrated through a graded series of alcohols
4. the alcohol is removed with xylene
5. a drop of cement followed by a cover slip is applied

Question 18

what is hematoxylin derived from

Answer 18

logwood as hematein

Question 19

what can orcein and resorcin fuchsin stains be used for?

Answer 19

to reveal elastic material

Question 20

what is silver impregnation useful for

Answer 20

to show reticular fibers and basement membranes

Question 21

how do acid dyes bind to tissue components?

Answer 21

by forming electrostatic linkages with cationic groups such as the amino groups of proteins

Question 22

define metachromasia

Answer 22

phenomenon whereby a dye changes color after reacting with a tissue component

Question 23

what can histochemical techniques be used for

Answer 23

to study the chemistry of cells and tissues

Question 24

what does the schiff reaction depend on

Answer 24

formation of aldehyde groups following exposure to HCL or periodic acid

Question 25

what is periodic acid used for

Answer 25

used to cleave bonds between adjacent carbons of carbohydrates and form aldehyde groups

Question 26

what is best carmine used for

Answer 26

dye that may be used to demonstrate glycogen deposits

Question 27

what can immunocytochemical techniques be used for

Answer 27

to study the presence of specific tissue constituents (antigens) by using monoclonal antibodies

Question 28

what are monoclonal antibodies derived from

Answer 28

activated B cell clones exposed to a specific antigen