Histology - Preparation of Cells (Steps)

How are cells prepared? How many steps, and in what order?

FDcESMs

1. Fixation

2. Dehydration & Clearing

3. Embedding

4. Sectioning

5. Mounting and Staining

Which step is Fixation? What occurs during this step?

1st Step

• Fermaldehyde/formalin

• Glutaraldehyde & Osmium tetroxide

• Prevents decay of tissue

Which step is Dehydration & Clearing? What occurs during this step? Is this step done at a different time, and if so what situation?

• Living tissues have a certain amount of water (makes preservation tricky)

  • Replace with ethanol or alcohols

  • Dunk this tissue in successively more concentrations of ethanol:water

• Done later for neural tissue

What step is Embedding? What occurs during this step, and why?

3rd step

• Material used: (Paraffin, resin/plastic, or Glycerol)

• Preventing impact (protect it)

• Tissue is very fragile (naturally soft)

• Frozen to keep them intact/harder

Which step is Sectioning? What occurs during this step? What products result from this step?

4th Step

• Sharp blade to cut thin sections of tissue

• Tissue is cut in very thin pieces to view under microscope

Which step is Mounting and Staining?

5th/Last step

• Mount = place on slides

• Stain with a substance (ex: Crystal Violet)

What tissue is this? Is there any issue with this image? What stain was mentioned in class?

Adipose tissue

• Currently Artifacted (white inside of cells shows that the fat was washed away

• Sudan red is able to stain the fat without washing it away

What is an artifact? Examples?

A structure on a microscoping slide that is not part of the normal tissue

• usually has an unnatural appearance

  • ex: lack of cellular detail, extremely reflective due to air bubble, etc.