Meghan Barboza, PhD
Online Course
Overview of course delivery
 Extremely accelerated. Look over the syllabus and
calendar. Plan accordingly.
 You are expected to spend at least 40 hours a week on
this course.
 You must have access to a PC (non-MAC) computer for
the lab software to work.
 You can work ahead. I will be posting
lectures/labs/quizzes as early as possible. You cannot
submit graded items after the date (11:59 pm) posted
on the calendar.
 E-mail is the best way to contact me.
barbozam3@southernct.edu
Another name for this
course: Microanatomy
Major Aims of Histology
 Understand a variety of tissue preparation and stains
Major Aims of Histology
 Relate the form of the tissue to its function:
Major Aims of Histology
 Recognize specific cell types and tissues in
healthy state
Eye (2011) 25,1–14
Major Aims of Histology
 Provide basis for recognizing abnormal state
Eye (2011) 25,1–14
Major Aims of Histology
 Appreciate differences between species
Major Aims of Histology
 Understand changes due to normal development
and aging
Histology: The Study of Tissues
Examination of
 cells: structure and function
 extracellular domain: interaction and
association of different cell types
and extracellular environment
Histotechniques
• Using a variety of tools to examine
cells and tissues includes:
• Fixation
• Preparation
Dehydration and Clearing*
• Embedding
•
• Sectioning
• Mounting and Staining
Predominant Technique for light
microscopy: Paraffin Sections
Plastic Sections
Frozen Sections
Flat Mounts
Electron Microscopy
 Scanning Electron Microscopy
(SEM)
 Transmission Electron
Microscopy (TEM)
http://egotvonline.com
How are slides prepared
 Samples collected and fixed
 Fixed samples are processed
 Samples are sectioned on a microtome or cryostat
 Stained according to:
 Tissue Type
 Question being asked
Fixation = Preservation
 Stop alterations of tissue (autolysis)
following death
 Maintain true to life architecture of tissue
 Most common fixative: Neutral Buffered
formalin (a type of formaldehyde)
 Other’s include:
 Paraformaldehyde (PFA)
 Glutaraldehyde - needed if doing EM
 Bouin’s
Objectives of a good fixative
Maintain true-to-life morphology
Allow variety of stains to be used
Budgetable
Dehydration and Clearing
 Paraffin sections
 Water is removed by washing tissue through a
series of alcohols
 Tissue undergoes clearing which uses the
chemical Xylene to fill in the areas previously
occupied by water and allows paraffin to
infiltrate.
Paraffin Sections
 Tissue is embedded in paraffin
 Sectioned at 5-6 microns
 Placed on a slide
 The section on the slide is
deparaffinized and stained
https://www.youtube.com/watch?v=KnMdSgd5mts
Paraffin Sections: Stains
 Hematoxylin and Eosin (H&E)
Paraffin Sections: Stains
 Hematoxylin and Eosin (H&E)
 Special Stains:

PAS (periodic acid-Schiff’s)
Paraffin Sections: Stains
 Hematoxylin and Eosin (H&E)
 Special Stains:
PAS (periodic acid-Schiff’s)
 Masson’s trichrome

Paraffin Sections: Stains
 Hematoxylin and Eosin (H&E)
 Special Stains:
PAS (periodic acid-Schiff’s)
 Masson trichrome
 Silver methenamine

Paraffin Sections: Stains
 Hematoxylin and Eosin
(H&E)
 Special Stains:
PAS (periodic acid-Schiff’s)
 Masson trichrome
 Silver methenamine
 Methylene Blue, Cresyl
Violet, etc…

www.vetmed.vt.edu
Summary of Stains (table 1.1)
Reagent
Result
Hematoxylin
Blue:nucleus, acidic cytoplasm, cartilage
matrix
Eosin
Pink:basic regions of cytoplasm,
collagen fibers
Masson’s trichrome
Dark blue: nuclei
Red: muscle, keratin, cytoplasm
Light blue: mucinogen, collagen
Orcein’s elastic stain
Brown: elastic fibers
Weigert’s elastic stain
Blue: elastic fibers
Silver stain
Black: reticular fibers
Iron hematoxylin
Black: striations of muscle, nuclei,
erythrocytes
Periodic acid-Schiff (PAS)
Magenta: glycogen and carbohydrate
rich (mucins)
Wright’s and Giemsa
Pink: erythrocytes
Blue: cytoplasm of
monocytes/lymphocytes
Paraffin Sections: Stains
 Hematoxylin and Eosin (H&E)
 Special Stains:
PAS (periodic acid-Schiff’s)
 Masson trichrome
 Silver methenamine
 Methylene Blue, Cresyl Violet, etc…
 Immunohistochemistry (IHC)

IHC
 Primarily the indirect method is
used.
 A primary antibody binds to the
antigen
 Non-bound antibody is washed
away
 A secondary antibody carries the
color (fluorescent or light)
Frozen sections
 In General:
 tissues are fixed or frozen
 embedded using a
special medium
 Sectioned on a cryostat
 Stained
 *no dehydration/clearing
https://www.youtube.com/watch?v=d43LFVV3h6w
Frozen Sections: Stains
 Many IHC’s don’t work on paraffin sections
 Oil Red O-a lipid stain
Common Problems
 Folds
Common Problems
 Folds
 Section tears
Common Problems
 Folds
 Section tears
 Uneven
staining
Common Problems
 Folds
 Section tears
 Uneven
staining
 Inadequate
Fixation
Check Your Understanding
 Within most lectures I will include these questions.
The answers to these will make up the majority of
your participation in the course. To participate go into
blackboard and create a thread with your answers to
the questions. You cannot view others answers or
respond until you have answered. Then go through
and read others responses.
 Here’s an example:
 To create the best end result, what is the most
important part of the process for creating histology
slides?
Questions