Fixation, Dehydration, Clearing

Question 1

It is important that tissues are handled carefully and appropriately fixed as soon as possible after arriving in the laboratory.

Answer 1

Fixation

Question 2

The specimen is placed in a liquid fixing agent (fixative) such as [?].

Answer 2

formaldehyde solution (formalin)

Question 3

This will slowly penetrate the tissue causing chemical and physical changes that will harden and preserve the tissue and protect it against subsequent processing steps.

Answer 3

liquid fixing agent (fixative)

Question 4

is the most popular fixative for preserving tissues that will be processed for paraffin embedding.

Answer 4

Formalin, usually as a phosphate-buffered solution

Question 5

Ideally, specimens should remain in fixative long enough for it to penetrate the tissue and then for an additional period in order to allow the chemical reactions of fixation to reach equilibrium (?).

Answer 5

fixation time

Question 6

is a critical step in the preparation of histological sections.

Answer 6

Fixation

Question 7

If it is not carried out under optimal conditions or if [?] is delayed, a tissue specimen can be irreversibly damaged.

Answer 7

Fixation

Question 8

Following fixation, appropriately trimmed specimens are placed in suitable labelled [?] (small perforated baskets) to segregate them from other specimens.

Answer 8

cassettes

Question 9

may be stored indefinitely at room temperature, and nucleic acids (both DNA and RNA) may be recovered from them decades after fixation.

Answer 9

Formalin-fixed, paraffin-embedded tissues

Question 10

Because melted paraffin wax is hydrophobic (not miscible with water), most of the water in a specimen must be removed before it can be infiltrated with wax.

Answer 10

Dehydration

Question 11

This process is commonly carried out by immersing specimens in a series of ethanol (alcohol) solutions with increasing concentration to avoid excessive distortion of tissue until a water-free tissue in alcohol is reached.

Answer 11

Dehydration

Question 12

Water soluble proteins are removed at lower concentrations of

Answer 12

ethanol.

Question 13

When ethanol concentration is increased to 100%, certain [?] may be dissolved.

Answer 13

lipids

Question 14

A typical dehydration sequence for specimens not more than 4mm thick would be:

Answer 14

70% ethanol 15 min
90% ethanol 15 min
100% ethanol 15 min
100% ethanol 15 min
100% ethanol 30 min
100% ethanol 45 min

Question 15

may be inadequately processed in what is normally a successful schedule for other tissues.

Answer 15

Fatty tissues such as breast or lipoma

Question 16

is a poor fat solvent.

Answer 16

Ethanol

Question 17

To ensure complete dehydration, a superior fat solvent such as [?] should be added before the [?], and [?] used as the transition solvent.

Answer 17

acetone or isopropanol

final absolute ethanol

chloroform or trichloroethane

Question 18

The dehydrated tissue is transferred to an intermediate solvent that is fully miscible with both

Answer 18

ethanol and paraffin wax.

Question 19

The term “clearing” has been chosen because many (but not all) clearing agents impart an [?] to the tissue due to their relatively high refractive index.

Answer 19

optical clarity or transparency

Question 20

Another important role of the clearing agent is to remove a substantial amount of fat from the tissue which otherwise presents a barrier to

Answer 20

wax infiltration.

Question 21

Following the dehydration, the tissue is immersed in one to three different [?] immersions.

Answer 21

xylene

Question 22

In these stages, the ethanol is gradually replaced with xylene and when the tissue is embedded, the xylene will then be replaced by the

Answer 22

molten paraffin wax.

Question 23

A typical clearing sequence for specimens not more than 4mm thick would be:

Answer 23

Xylene 20 min
Xylene 20 min
Xylene 45 min