Part 3

Question 1

Is a well-known artifact that may be produced under acid conditions.

Answer 1

Formalin pigment

Question 2

May be found in surgical specimens particularly in liver biopsies, associated with an intense eosinophilic staining at the center of the tissue in H&E stained sections.

Answer 2

Crush artifact

Question 3

Should be used for demonstrating lipid in tissues, followed by a general lipid stain

Answer 3

Cryostat or frozen sections

Question 4

May be used to preserve phospholipids

Answer 4

Baker’s Formal-Calcium

Question 5

Cholesterol may be fixed with __ for ultrastructural demonstration

Answer 5

Digitonin

Question 6

Generally recommended for glycogen fixation

Answer 6

Alcoholic fixatives

Question 7

Is a better fixative in human skin compared with neutral buffered formaldehyde.

Answer 7

Alcoholic formaldehyde

Question 8

The most useful fixatives for preserving glycogen are alcohol-based

Answer 8

Rossman’s fuid or cold absolute alcohol

Question 9

Are the most commonly used fixatives for amino acid histochemistry.

Answer 9

Neutral buffered formal saline or formaldehyde

Question 10

The most useful primary fixatives for electron microscopy are

Answer 10

Osmium tetroxide, glutaraldehyde and paraformaldehyde

Question 11

General aim of fixation for enzyme histochemistry

Answer 11

Preserve maximum enzyme activity at its original localization while also preserving structural integrity

Question 12

Are commonly used in pathology for the demonstration of various antibodies.

Answer 12

Immunofluorescence

Question 13

Are required for successful immunohistochemistry.

Answer 13

Antigen-retrieval techniques

Question 14

Solvents such as alcohols and acetone remove lipids and dehydrate the cells, while precipitating the proteins on the cellular

Answer 14

Organic Solvents

Question 15

Form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens.

Answer 15

Cross-linking reagents

Question 16

T/F: Cross-linkers preserve cell structure better than organic solvents, but may reduce the antigenicity of some cell components, and require the addition of a permeabilization step, to allow access of the antibody to the specimen.

Answer 16

TRUE

Question 17

Causes covalent cross-links between molecules, effectively gluing them together into an insoluble meshwork

Answer 17

Paraformaldehyde

Question 18

This will avoid or reduce the ‘auto fluorescence’ (i.e., high background) that is inherent to formalin fixed paraffin embedded tissue when performing immunofluorescent microscopy.

Answer 18

Cryopreservation

Question 19

This will avoid or reduce the ‘auto fluorescence’ (i.e., high background) that is inherent to formalin fixed paraffin embedded tissue when performing immunofluorescent microscopy.

Answer 19

Cryopreservation

Question 20

This stain require frozen sections since the ethanols used in tissue processing will extract the lipids.

Answer 20

Lipid stains (Oil red O)

Question 21

best done on frozen sections

Answer 21

Immunofluorescent staining (IF)

Question 21

Fix cells in -20°C acetone for 5-10 minutes.
No permeabilization step needed following acetone fixation

Answer 21

Acetone

Question 22

Fix cells in -20°C methanol for 5-10 minutes. Permeabilization step is needed following methanol fixation.

Answer 22

Methanol fixation

Question 23

Fix cells in cooled 95% ethanol, 5% glacial acetic acid for 5-10 minutes.

Answer 23

Ethanol fixation

Question 24

Fix in cooled methanol, 10 minutes at –20 °C.
Remove excess methanol.
Permeabilize with cooled acetone for 1 minute at –20 °C.

Answer 24

Methanol-Acetone fixation

Question 25

1:1 methanol and acetone mixture.
Make the mixture fresh and fix cells at -20 C for 5-10 minutes.

Answer 25

Methanol-acetone Mix Fixation

Question 26

1:1 methanol and ethanol mixture.
Make the mixture fresh and fix cells at -20 C for 5-10 minutes.

Answer 26

Methanol-Ethanol Mix Fixation

Question 27

Fix cells in 10% neutral buffered formalin for 5-10 minutes. Rinse briefly with PBS.
Permeabilize with 0.5% Triton X-100 for 10 minutes.

Answer 27

Formalin Fixation

Question 28

Fix in 3-4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS.
Permeabilize with 0.5% Triton X-100 for 10 minutes.

Answer 28

Paraformaldehyde-Triton Fixation

Question 29

Fix in 4% paraformaldehyde for 10-20 minutes.
Rinse briefly with PBS.
Permeabilize with cooled methanol for 5-10 minutes at –20 °C.

Answer 29

Paraformaldehyde-Methanol Fixation

Question 30

T/F: Do not reuse or save diluted antibodies. The antibodies adhere to the walls of the container and this effectively reduces the titer to zero.

Answer 30

TRUE

Question 31

T/F: Freeze / thaw cycles will destroy antibodies.

Answer 31

TRUE

Question 32

Have shown that some of the reactions of fixation are reversible, particularly those of formaldehyde, but there is considerable variation in the quality of antigen preservation with various agents.

Answer 32

Antigen-retrieval methods in immunohistochemistry

Question 33

Microwave antigen retrieval is one of several so-called

Answer 33

HIER (Heat Induced Epitope Retrieval)

Question 34

Temperatures of between _ and _ are commonly employed.

Answer 34

37degC and 45degC

Question 35

Characterized by an exaggerated and different morphological and staining characteristics on the outside compared to the inside of the cell

Answer 35

“zonal” fixation effect

Question 36

Allows light microscopic techniques used in routine histopathology to be performed adequately.

Answer 36

Microwave fixation

Question 37

Considered to be the major factor responsible for the effects of microwaves during tissue fixation

Answer 37

Heat

Question 38

T/F: After microwaving they should immediately be sliced to 2 mm and placed in 70% ethanol.

Answer 38

TRUE

Question 39

For microwave assisted fixation, __ thick slices should be prepared from tissue

Answer 39

2mm

Question 40

Advantages of Microwave Fixation

Answer 40

1. The chief advantage of microwave fixation is that the tissue is heated right through the block in a very short time, thereby potentially allowing the study of cellular processes that proceed very rapidly.
2. It is a non-chemical technique that is useful in preserving neurochemical substances in brain, such acetylcholine.
3. It may also be used for the rapid fixation of routine surgical specimens.
4. It significantly reduces the time taken for immuno-histochemistry and in-
   situ hybridization.
5. Microwave ​ treated tissue (at 50oC), post-fixed in osmium tetroxide also
   gives satisfactory results for electron microscopy.