Histology & Its Methods of Study: Enzyme (Immuno)Histochemistry

Question 1

define enzyme histochemistry

Answer 1

Histochemical (or cytochemical) techniques using specific enzymatic activities in lightly fixed or unfixed tissue sections to produce visible products in the specific enzyme locations.

Question 2

Because Fixation and paraffin embedding denatures most enzymes, histochemistry usually use…

Answer 2

frozen tissue sectioned with a cryostat

Question 3

state the 4 steps of enzyme histochemistry

Answer 3

1. sections immersed in solution with enzyme substrate to be localized
2. enzyme acts on substrate
3. section put in contact with marker compound that reacts with product of enzymatic action on substrate
4. final marker product (insoluble and visible via light or electron microscopy) precipitates over the sites of the enzymes, identifying their location

Question 4

give examples of enzymes that can be detected histochemically

Answer 4

phosphatases - remove phosphate groups from macromolecule

dehydrogenases - transfer hydrogen ions from one substrate to other, (like enzymes in Krebs cycle) allowing identification of such enzymes in mitochondria

peroxidase: promotes oxidation of substrates with transfer of hydrogen ions to hydrogen peroxide

• ex: Isocitrate Dehydrogenase: Converts isocitrate to α-ketoglutarate, producing NADH.

α-Ketoglutarate Dehydrogenase: Converts α-ketoglutarate to succinyl-CoA, producing NADH.

Succinate Dehydrogenase: Converts succinate to fumarate, producing FADH2.

Malate Dehydrogenase: Converts malate to oxaloacetate, producing NADH.

Question 5

Visualizing Specific Molecules

Answer 5

Some substances specifically bind certain targets in cells.

• ex: lectins, glycoproteins derived from plant seeds with high affinity and specificity for carbohydrates, sugars, sugar residues, and allow fluorescently labeled lectins to stain specific glycoproteins

Question 6

define Immunohistochemistry

Answer 6

highly specific interaction between antigen and its antibody where antibodies are used to identify and localize specific proteins
- based on specific reactions between an antigen and antibodies labeled with visible markers, often fluorescent compounds or peroxidase for light microscopy and gold particles for TEM.

Question 7

what family do antibodies belong to?

Answer 7

the immunoglobin family of glycoproteins secreted by lymphocytes

Question 8

What is direct immunocytochemistry?

Answer 8

when the cell or tissue antigen of interest is detected by directly binding a labeled primary antibody specific for that antigen

Question 9

what is Indirect immunohistochemistry?

Answer 9

unlabeled primary antibody that is detected bound to its antigen with labeled secondary antibodies.

The indirect immunohistochemical method is more commonly used because the added level of antibody binding amplifies the signal detected and provides greater technical flexibility.

Question 10

What is in situ hybridization (ISH)?

Answer 10

Specific gene sequences or mRNAs of cells can be detected microscopically using labeled cDNA probes in a technique called in situ hybridization (ISH)

Question 11

state the 3 steps of ISH

Answer 11

1. determining specific sequence DNA of cell
2. identifying mRNA-containing cells
3. determining location of gene on specific chromosome

• specific nucleotide sequences are detected with probes consisting of single-stranded complementary DNA (cDNA)