1. LESSON 7
FIXATION

2. If you want a permanent sample,
FIXATION !

3. Why Fixation?
To avoid self-destruction (or autolysis) by intracellular enzymes
To prevent bacterial decomposition
To preserve the structure and molecular composition

4. the first step and the foundation in a sequence of histology

5. Fixation
The technique in the preparation of specimens for the purpose of maintaining the existing form and structure of all the constituent elements.
Chemical fixation
Physical fixation

6. Fixation
The technique in the preparation of specimens for the purpose of maintaining the existing form and structure of all the constituent elements.
Chemical fixation
makes cells permeable to staining reagents and crosslinks their macromolecules so that they are stabilized and locked in position using fixatives .
Physical fixation

7. The ideal/best fixation ……
Preservation in a condition identical to that existing during life.
a life-like state as possible!
Promptly (ASAP) and adequately

8. Intravascular Perfusion
Facilitate the penetration of the fixative

9. Fixation Safe guard against the damaging
To facilitate differential staining with dyes and other reagents.

10. Fixatives Organic solvents Heavy metal fixatives Alcohol Aldehyde
precipitate proteins and other macromolecules.
Aldehyde
chemically cross-links the primary amines of neighboring proteins
Heavy metal fixatives

11. The most common fixatives for routine light microscopy ?
“Formalin”
A buffered isotonic solution of % formaldehyde (by weight)

12. Formaldehyde HCHO causes proteins to cross link in a meshwork
Lipids, nucleic acids and carbohydrates are trapped in a matrix of insoluble and cross-linked proteins
milder fixative

13. Formaldehyde's mechanism of action
(within 24 hr)
(several weeks)
In practical terms, formaldehyde penetrates tissues rapidly (because of its small size), but it slowly cross-links the proteins.

14. Methylene bridges between the side amino groups

15. Formaldehyde HCHO causes proteins to cross link in a meshwork
milder fixative
Formalin
Paraformaldehyde
Higher polymers (n up to 100) are insoluble in water and sold as a white powder

16. Formation of formaldehyde polymers
Depolymerization of paraformaldehyde
To be useful as a fixative, the solution must contain monomeric formaldehyde.

17. +10-14% MeOH as stabilizer, inhibit polymerization
1 in 10 dilution

18. 2. Glutaraldehyde HCO-[CH2]3-CHO
enormous potential for cross-linking proteins
standard fixative for EM
'EM grade' glutaraldehyde

19. 2. Glutaraldehyde HCO-[CH2]3-CHO
Polymerization reaction of glutaraldehyde
In solution glutaraldehyde exists as polymers of various sizes which exhibit an enormous potential for cross-linking proteins

20. Reaction of polyglutaraldehyde with amino groups of proteins.
In contrast with formaldehyde, the chemical reaction of glutaraldehyde with protein is fast, but the penetration of tissue is extremly slower, especially for the larger oligomers.
(fast)

21. formaldehyde vs. glutaraldehyde
Fixation (cross-linking)
Penetration

22. formaldehyde and glutaraldehyde
The combination takes advantage of the rapid penetration of formaldehyde, which quickly stabilizes the structure of the tissue, followed by a more thorough cross-linking of proteins mediated by the more slowly penetrating glutaraldehyde
2-4 % paraformaldehyde % glutaraldehyde

23. 3. Osmium Tetroxide (OsO4)
fixative & stain
binds to and stabilizes lipid as well as proteins
Secondary fixative
OsO4 → OsO4∙nH2O (osmium black)

25. Double fixation Standard procedure for Electron Microscopy
1st nd
Special Preparation for fine structural studies

27. 4. Ethanol C2H5OH Coagulates protein (but not nucleoprotein)
Precipitates glycogen
Dissolves many lipids
Powerful dehydrating agent
Shrinkage & hardening

28. 5. Acetic acid pure water-free CH3COOH ‘glacial acetic acid’
Used in many fixing mixtures
Precipitate nucleoprotein

29. 6. cold Methanol or Acetone
Rapid fixation (than aldehydes)
Precipitating proteins and carbohydrates
Fixation and dehydration occur simultaneously
Possible shrinkage of the samples

30. Factors involved in fixation
Temperature .
Size of specimens and penetration of fixatives.
Volume of fixatives (10 times that of the tissue).
pH and buffers .
Osmolality .
Concentration of fixatives .
Duration of fixation
slow (by cold) / 37℃ (accelerated) / heat (almost inhibit, but …..)

31. Learning Resources Junqueira’s Basic Histology ; pp 1-3
Looking at the Structure of Cells in the Microscope
“Tissues Are Usually Fixed and Sectioned for Microscopy.”

32. The Theory & Practice of Staining
Lesson 8 Staining
The Theory & Practice of Staining

33. Why staining ? Most cells and tissues are transparent and colorless.
One way to make them visible is to stain them with dyes.
Successful staining ?
Specificity - selectivity, the ability to discriminate between individual tissue component
Sensitivity – a capacity of the stain to demonstrate a tissue substance in low concentration

34. Types of staining Vital staining Elective solubility
Staining with dyes
Metal impregnation
Production of colored substances

35. Types of staining Vital staining Elective solubility
In living tissues/cells
trypan blue (cytoplasmic phagocytosis)
Janus green (mitochondria)
Elective solubility
Staining with dyes
Metal impregnation
Production of colored substances

36. Types of staining Vital staining Staining by Elective Solubility
Lipid-rich structures (e.g. fat droplets)
Lipid-soluble dyes (lipophilic dye)
Frozen sections
Staining with dyes
Metal impregnation
Production of colored substances

37. Sudan black
stained the lipid droplets & adipose cells

38. Types of staining Vital staining Elective solubility
Staining with dyes
Basic & Acidic dyes
Metachromatic Staining
Local formation of a dye
Metal impregnation
Production of colored substances

39. Basic & Acidic dyes Basic /cationic dye Acidic/anionic dye
Basophilic (e.g. nucleic acids, glycosaminoglycans, acid glycoprotein & other acidic structures)
affinity for negatively charged molecules
Toluidine blue, alcian blue, methylene blue, Hematoxylin
Acidic/anionic dye
Acidophilic (e.g. proteins with many ionized amino groups, mitochondria, secretory granules, collagen)
Orange G, eosin, acid fuchsin
No relevance to pH
Positive charge (cationic) / Negative charge (anionic)

40. H&E (Hematoxylin and Eosin)
Hematoxylin (blue-purple)
DNA of the cell nucleus, RNA-rich portion
Eosin (pink)
Other cytoplasm, collagen
Good Counterstain
a stain with color contrasting to the principal stain, making the stained structure more easily visible

41. PAS with H&E staining

42. General procedure Deparaffin (Re)Hydration Hematoxylin Rinse in water
Eosin
Dehydration
Xylene
Mounting
a protective glass coverslip on the slide with adhesive mounting media (Canada Balsam, Gelvatol……)

43. Metachromatic Staining
Metachromasia
To produce a color other than that of the dye used
Metachromatic tissue
Cartilage, connective tissue, amyloid ……
Metachromatic dye
Toluidine Blue
orthochromatic color
nucleic acids blue
metachromatic color
sulfated polysaccharides purple

44. Local formation of a dye
Feulgen stain
DNA
Periodic acid-Schiff(PAS) stain
carbohydrate   

45. Feulgen stain DNA can be specifically identified and quantified
Mild hydrolysis with HCl frees the aldehyde group of deoxyribose in DNA, which is then reacted with the Schiff's reagent.

46. Periodic acid-Schiff(PAS) stain
for staining structures containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans)
Periodic acid
Schiff reagent
an aqueous solution of rosaniline and sulfurous acid
iodic(VII) acid, HIO4 or H5IO6

47. Periodic acid-Schiff(PAS) stain
periodic acid is to oxidize some of the tissue carbohydrates. This produces aldehyde groups,
which can then condense with Schiff's reagent forming a bright purple or magenta coloration.
+  HIO4  =  
+ 2(R-CHO) =

48. Types of staining Vital staining Elective solubility
Staining with dyes
Metal impregnation
e.g. silver staining
A common method of visualizing certain ECM fibers & specific cellular elements in nervous tissue
Production of colored substances

49. Learning Resources Junqueira’s Basic Histology ; pp 3-4
Looking at the Structure of Cells in the Microscope
“Different Components of the Cell Can Be Selectively Stained ”

51. LESSON 9. HISTOCHEMISTRY & CYTOCHEMISTRY
كيمياء خلايا وأنسجة الجسم البشري
الكيمياء الخلوية

52. Lecture Overview What’s histochemistry / cytochemistry ?
Principle (Enzyme Reaction)
Procedure
Examples

53. Histochemistry & Cytochemistry
Staining
Enzyme histochemistry
Immunohistochemistry

54. Enzyme histochemistry
Methods for localizing cellular structures using unique enzymatic activity present in those cell structures.
e.g.
Lysosome : acid phosphatase
Membrane : alkaline phosphatase
Mitochondria : dehydrogenase

55. Enzyme Reaction Substrate: the substance acted upon by an enzyme
Speed up
Substrate: the substance acted upon by an enzyme

56. Preparation of tissues for enzyme techniques
To preserve enzymatic activity
Fresh unfixed or mildly fixed tissue
Rapid disappearance of most enzymes after death
Cryostat
Speed, ease of handling, and versatility

57. Procedure Incubate in Substrate solution
Enzymatic action on the substrate
Formation of primary reaction product (PRP)
Contact with a Marker compound (Trapping agent)
Reacts with a PRP
contains a visible reaction product such as a heavy metal or a colored reaction product.
Final reaction product (FRP)
Insoluble
Visible by microscopy
Color, electron-dense material, precipitates
Trapping agent - binds to the substrate and contains a visible reaction product such as a heavy metal or a colored reaction product.

58. Types of Methods Simultaneous capture Post-incubation coupling
Production of the PRP and its capture as a coloured FRP take place simultaneously in one incubating medium
Post-incubation coupling
Two reactions are carried out separately one after another
Production of a colourless PRP first
Subsequently coupled into a coloured FRP in a separate solution.

59. Phosphatases Alkaline phosphatase Acid phosphatase
: removes a phosphate group from its substrate
Alkaline phosphatase
Acid phosphatase
pH 9.0
Kidney, intestine, ……
≈pH 5.0

60. Dehydrogenases mitochondria
Remove hydrogen from a substrate and transfer it to a hydrogen receptor
mitochondria

61. Peroxidase myeloperoxidase
Promotes the oxidation of certain substrates with the transfer of hydrogen ions to hydrogen peroxide, forming molecules of water
myeloperoxidase
Diagnosis of certain leukemia (cancer of blood)