1. Topic 1: Introduction to Histology
Animal Histology BIOL 241
Topic 1:
Introduction to Histology
Dr. Issa Al-Amri
Department of Biological Sciences & Chemistry
College of Arts & Sciences

2. Histological Techniques
Methodology (Histological Techniques):
Type of sample preparation methods:
Preparation methods for light microscopy
To prepare tissue for LM examination, fixed tissues used. Samples of fixed tissues are used in the form of:
Sections on glass slides.
Smears on glass slides for blood, mucus, urine, and bone marrow.
Spreads for loose (areolar) connective tissue.
Teased samples for muscle tissue (individual fibres).

3. Histological Techniques
Tissue processing:
Removal of specimen: tissue specimen dissected from the animal and placed in fixative.
Fixation: fixing fluids (buffered 10% formaldehyde) preserve structures (cross-link proteins) and inhibit autolysis (digestion by enzymes) and bacterial growth.
Dehydration & clearing: dehydration is removal of water from tissue and replace it with graded conc. of ethanol. Clearing is replacing ethanol with solvent (xylene) that mix with paraffin wax. Process carried in tissue processor.
Infiltration: gradual replacement of xylene with wax (60C)
Embedding: tissue embedded in paraffin provides rigid support to tissue blocks to be cut into thin sections. Embedding station used for tissue orientation and wax.

4. Histological Techniques
Fixation of animals (formalin)

5. Histological Techniques
Formalin fixed brain

6. Histological Techniques
Tissue dissected and placed in cassette for processing

7. Histological Techniques
Automatic Tissue Processer

8. Histological Techniques
Tissue Embedding Station

9. Histological Techniques
Tissue embedding process – processed tissue removed from cassette

10. Histological Techniques
Tissue embedding process – tissue oriented in a mould

11. Histological Techniques
Tissue embedding process – paraffin added to fill cassette and mould

12. Histological Techniques
Tissue embedding process – wax solidification in cold plate

13. Histological Techniques
Tissue Blocks ready for sectioning

14. Histological Techniques
Sectioning (microtomy):
The tissue block then cut into thin sections µm with a rotary microtome.
Sections then mounted on glass slides and stained.
Frozen sections obtained after tissue fixation by freezing, then cut into sections by cryostat (freezing microtome), mounted on glass slides and stained. This method used in hospitals for quick diagnosis during surgery and to study the enzymes by histochemical methods.

15. Histological Techniques
Rotary Microtome

16. Histological Techniques
Sectioning processes

17. Histological Techniques
Ribbon of Paraffin Sections into water bath

18. Histological TechniquesB
Ribbon of Paraffin Sections in water bath. A. Sections with wrinkles. B. Sections stretched

19. Histological Techniques
Paraffin Sections picked up in glass slide

20. Histological Techniques
Sections in glass slide left to dry in hot plate

21. Histological Techniques
Histological Staining
To stain tissue and make some parts appears darker than others is by use of dyes which stain tissue components selectively. Dyes are either acidic or basic.
Tissue structures stained with basic dyes such as haematoxylin, methylene blue, and toluidine blue, are said to be basophilic.
Tissue structures stained with acidic dyes such as eosin, is said to be acidophilic.

22. Histological Techniques
Histological Stains
Routine H&E stain:
Routine staining of tissue usually employs Haematoxylin (H) and Eosin (E) stains to stain general structures.
Haematoxylin reacts with nucleic acids and acidic groups of proteins and stain them blue. The nucleic acids are concentrated in the nucleus and rough endoplasmic reticulum (RER).
Eosin, stains the cytoplasm of most cells red or pink. The cytoplasm may be basophilic, acidophilic or neutrophilic.

23. Histological Techniques
- Harris’s H&E Staining Procedure:
Sections (in slides) deparaffinised in xylene.
Sections then hydrated through graded ethanol concentration, and then washed in running tap water.
Slides then stained with Harris’s haematoxylin, then washed in running tap water.
Slides then differentiated in 1% acid alcohol, and rinsed in running tap water.
Slides taken into blueing solution (weak ammonia), then rinsed in running tap water.
Slides stained in 1% aqueous eosin Y, then rinsed in tap water.
Slides dehydrated through graded alcohol concentration, and cleared through a series of xylene changes.
Slides then mounted in D.P.X. , covered with coverslip.

24. Histological Techniques
Manual staining

25. Histological Techniques
Automatic staining

26. Sections stained with H & E stain, mounted with D.P.X , covered
Histological Techniques
Sections stained with H & E stain, mounted with D.P.X , covered
with coverslips

27. Histological Techniques
Kidney glomerulus stained with H & E stain

28. Digital light microscopy: imaging using digital camera, computer
with camera program and a monitor

29. Histological Techniques
Histological Staining
Special stains:
Used to stain special structures such as glycogen granules and fibrils.
Counter stain (e.g. Mayer’s haematoxylin) is then used to reveal nuclei and the background of special structures.
Trichrome stains such as Mallory and Masson`s stains applied to differentiate between collagen and smooth muscle fibres.

30. Histological Techniques
Histochemical and cytochemical staining:
Methods used to localize substances in tissue sections based on specific chemical reactions between macromolecules and production of insoluble coloured substances seen under microscope.
Examples of studied substances:
Ions: such as calcium, iron, and phosphate.
b. Nucleic acids: DNA and RNA studied by use of basic dyes.

31. Histological Techniques
Proteins: study of enzyme activity in tissue by localising the action of enzyme with its substrate by producing coloured insoluble substance that can be seen under the microscope.
The most common enzymes studied:
Phosphatase: acid phosphatase (lysosomes in macrophages) and alkaline phosphatase (liver).
Dehydrogenase: succenic dehydrogenase of mitochondria.
Peroxidase: present in liver and kidney cells; it promotes oxidation of its substrates.

32. Histological Techniques
d. Polysaccharides and oligosaccariades: carbohydrates present either free (glycogen) or as combined with lipids as glycolipids or with proteins as glycoproteins. Carbohydrate part can be demonstrated by use of PAS reaction.
PAS (Periodic Acid Schiff reagent): method used to stain glycosamino-glycan [GAG], glycoproteins, and glycogen which then counterstained by hematoxylin.
e. Lipids: lipids dissolve in routine preparations, best to use dyes that dissolve in lipids. Frozen sections dipped into alcohol saturated with Sudan IV or Sudan black which stains lipid droplets red or black respectively.

33. Histological Techniques
Common Stains in Light Microscopy:
H & E: routine; stains nuclei blue and the cytoplasm pink.
Azocarmine: used as a counter stain. Stains glycogen and mucigen.
Mallory-Azan: stains nuclei red and the cytoplasm orange or blue.
Masson`s trichrome: stains nuclei black or dark blue, cytoplasm stains red, reticular and collagen fibres stain blue.
Periodic acid-Schiff`s and hematoxylene (PASH): stains glycogen, mucin and carbohydrate containing tissue red or pink and the nuclei blue.
Silver stain: silver nitrate used to stain reticular fibers, neurofibrils and granules in enteroendocrine cells.
Orcein stain: for elastic fibres.

34. Histological Techniques
Thionin stain: stain Nissl`s granules blue in nerve cells.
Toluidine blue: stains granules in mast cells reddish-purple, while nuclei stained deep blue.
Trypan blue: vital stain living cells in vivo, or supra vital stain living cells in vitro. In vivo, stain injected into living animals to demonstrate phagocytic cells which ingest stain granules.
Verhoff`s stain: stains elastic fibres black.
Osmium tetroxide: stain lipid contents of cells black colour e.g. myelin sheath of nerve fibres.
Sudan III: stains lipids with orange colour e.g. fat droplets in adipocytes.

35. Histological Techniques

36. Histological Techniques
PAS-Fast Green. Uterine tube. cytoplasm of nonciliated cells (NC) contains secretory
granules (SG).

37. Histological Techniques
Masson’s Trichrome stain showing glomerulus with slight
mesangial prominance.

38. Histological Techniques
Masson’s Trichrome stain of muscular artery : Collagen and nerve cells stained blue. Muscle,
elastic fibres and blood stained red. Cytoplasm of cells stained red. Nuclei stained purple.

39. Histological Techniques
Mallory-Azan stain of fibres

40. Histological Techniques
Immunohistochemistry:
Highly specific reaction between an antigen (foreign protein) and its antibody (immunoglobulin).
Tissue sections incubated with antibodies to their contents of proteins so each antibody binds specifically to its protein (antigen) denoting the protein location in the cell that can be seen under the microscope.
Polyclonal antibodies: antibodies produced and collected contain mixture of several groups of antibodies for different parts of proteins (antigens).
Monoclonal antibodies: antibodies for different parts of proteins are collected separately.

41. Histological Techniques
Localization of IgG4 (red) in mouse Pancreas

42. Histological Techniques
Alzheimer :Tau in Neurofibrillary tangles in human brain

43. stained positive in nuclei and cytoplasm (arrow).
Histological Techniques
Immunolocalization of previtellogenic follicle PRs. All granulosa cells
stained positive in nuclei and cytoplasm (arrow).