E1: Cell & tissue type basics

Question 1

Describe the steps in FFPE tissue biopsy prep

Answer 1

1. Fixation:
   
   1. Add formalin to inactivate enzymes & prevent the tissue from quickly autolyzing
2. Dehydration:
   
   1. Replace water w/ ethanol since Paraffin is hydrophobic and needs to infiltrate the specimen
3. Clearing:
   
   1. Replace ethanol w/ Xylene or Toluene
4. Embedding:
   
   1. Infiltrate w/ molten paraffin wax
   2. Prevents distortion when cutting the soft tissue
5. Sectioning:
   
   1. Cut on a microtome
   2. Small enough to allow light to pass through
6. Staining:
   
   1. Stain w/ Hematoxylin & Eosin to give contrast to the specimen

Question 2

Describe the steps in Frozen Section biopsy prep.

What are the advantages of this process over FFPE?

Answer 2

1. Tissue is fixed & hardened in LN2
2. Sectioned in Cryostat
3. Stained & examined by pathologist

Frozen sections are faster and can be reported back in real time to a surgeon if necessary.

Question 3

What are the dyes used most commonly in staining?

What do they bind to & why?

Answer 3

H&E = Hematoxylin & Eosin

Hematoxylin is a positively charged molecule with a Blue appearance & preferentially binds to negatively charged (basophilic) molecules (i.e. DNA, RNA, & sulfated polysaccharides)

Eosin is a negatively charged molecule with a Pink appearance and preferentially binds to positively charged (acidophilic) molecules (Proteins)