Help one help everyone :)

2. DECALCIFICATION
SHRAVYA.M

3. CONTENTS
CONTENTS
• Introduction
• Biopsy
• Criteria of a good decalcifying agent
• Factors affecting decalcification
• Technique
• Bone Decalcification
• Teeth Decalcification Selection
Processing
Staining
• Endpoint Decalcification
• Undecalcified Sections Of Bone
• Properitary Decalcifiers
• Artefacts
• Recent Studies
• Recent Advances
• References

4. INTRODUCTION
 To study the histological structure, the tissue should be
appropriately prepared for microscopic examination.
 Tissue specimens must be thin enough to permit the passage of
light & should be one cell thickness for detailed morphology.

5. Average thickness is 4-6 µm.
To section the hard mineralised tissue
decalcification is necessary

6. BIOPSY
• A biopsy is a procedure performed to remove tissue or
cells from the body for examination under a
microscope.
BONE BIOPSY
• A bone biopsy is a procedure in which a small sample
of bone is taken from the body and looked at under a
microscope for cancer, infection, or other bone
disorders.

7. Decalcification is a routine procedure with the
purpose of making a calcified tissue compatible
with the embedding media for cutting micro
slides and subsequent staining.

8. Criteria of a good decalcifying agent :
1. Complete removal of calcium
2. Absence of damage to tissue cells or fibres
3. Non impairment of subsequent staining technique
4. Reasonable speed of decalcification

9. FACTORS AFFECTING THE RATE OF
DECALCIFICATION:
Concentration of decalcifying agent
• Large volume of the fluid compared with the volume of tissue- 20 to 1 is
recommended to avoid total depletion of the acid or chelator by their
reaction with calcium.
• Fluid should be changed several times during the decalcification process
Temperature
• Increased temperature accelerates decalcification but also increases the
damaging effects of acids on tissue. 18º C -30º C is acceptable.

10. Agitation
Gentle agitation may increase the rate slightly by influencing
fluid exchange within as well as around tissues.
Suspension
Fresh decalcifier should have ready access to all surfaces of the
specimen. enhance diffusion and penetration into the specimen
and facilitate solution, ionization and removal of calcium.

11. TECHNIQUE
The technique of decalcification is divided into the
following stages:
1.Selection of tissue
2.Fixation
3.Decalcification
4.Acid neutralization &
5. Thorough washing

12. SELECTION OF THE TISSUE
BONE / TEETH
Fine toothed bone saw or hack saw(large sp)
Geological cutting machine fitted with a
diamond impregnated cutting disc (small sp)
Slices not exceed 4-5mm in thickness.

13. FIXATION
• As a routine fixative, formal-saline is preferred but bone marrow is best
fixed in Zenker formol.
• For tooth specimens , 15% formic acid is mostly preferred
• For electron microscopy – Gluteraldehyde
• Some fine preparations of bone have been produced following immersion in
Mullers fluid followed by decalcification in 3% formic acid – formalin.

14. Tissue damage during acid
decalcification is four times greater when
the tissue is unfixed.

15. DECALCIFICATION
Decalcification is the process of removing inorganic calcium
(mineral) content of the bone /tissue before processing the
specimen after fixation.
Choice of decalcifying agent influenced
Urgency of the case
Degree of mineralization
Extent of investigation
Staining technique required

16. Routine decalcification methods include:
1. Acids
2. Ion exchange resins
3. Electrical ionization
4. Histochemical methods
 Buffer mixtures
 Chelating agents
5. Surface decalcification

17. ACID DECALCIFICATION
Principle
Acid releases calcium from its chemical combinations with
phosphates and carbonates in bone through ionic
exchange giving soluble calcium salt.
Two types
• Strong Inorganic Acids
• Weak Organic Acids

18. Strong acids
• It Is An Inorganic Acid
Eg: Nitric Acid, Hydrochloric Acid
• Recommended Concentration - 5-10%
• They Decalcify Rapidly By Dissolving
Calcium

19. MINERAL ACID DECALCIFIERS

20. a)Nitric acid
Nitric acid 5-10ml
Distilled water 100ml
1.Fix the selected block of bone for 2-3 days in buffered neutral formalin.
2.Place a mixture of 95ml distilled water and 5ml of nitric acid.
3.Change nitric acid solutions daily until bubbles cease to evolve from
the tissues(1-3 days,depending on the size and consistency of the bone
block)
4.Wash in 3 changes of 90% alcohol.
5.Dehydrate,clear in xylene or benzene and embed in paraffin

21. Formation of nitrous acid checked temporarily by
addition of 0.1% urea to the conc nitric acid
It’s the fastest decalcifier, but end point must be
carefully watched .
Yellow discolouration owing to formation of
nitrous acid, this accelerates decalcification but
also stains and damage tissues

22. Advantages
Rapid in action
Gives better nuclear staining
Causes very little hydrolysis
For Needle & Small Biopsy Specimens To Permit
Rapid Diagnosis .
Disadvantages
Tissue left for long time causes damage to tissue
Urea is added to remove yellow color of tissue

23. b) PERENYI’S FLUID (PERENYI 1882)
10% Nitric Acid 40ml
Absolute Ethanol 30ml
0.5% Chromic Acid 30ml
 Mix Shortly Before Use
 Chromic Acid Must Be Collected For Proper Disposal.
 Its Popular Especially For Small Specimens That Are
Not Densely Decalcifed

24. NOTE
•Strong acids are more damaging to
 tissue antigens for immunohistochemical staining
 enzymes may be completely lost.
•Strong acids are used for needle & small biopsy specimens to permit rapid diagnosis
within 24 hours.

25. WEAK ACID DECALCIFIERS
ACETIC ACID
PICRIC ACID

26. Weak, organic acids
e.g. formic, acetic, picric.
•Acetic & picric acid cause tissue swelling & are not used alone
as primary decalcifiers but are found as components of Carnoy’s
& Bouin’s fixatives

27. • Formic acid is the only weak acid used extensively
as a decalcifier
•Formic acid solutions are either
•aqueous (5-10%)
•buffered or combined with formalin.

28. The formalin/10% formic acid mixture simultaneously fixes & decalcifies.
• Recommended for –
• Very small bone pieces
• Jamshidi needle biopsies.
• Formic acid gentle & slower than Hcl or nitric acid
• suitable for most routine surgical specimens, particularly for immuno
histochemistry.
• Decalcification usually complete within 2-7days.

29. a)AQUEOUS FORMIC ACID
1.Well fixed 2-5mm thick blocks are placed in
– concentrated formic acid 5-25ml
– Distilled water 100ml
– 40% formaldehyde (optional) 5 ml
2.Change daily until decalcification is complete.
( 1-7 days for an average blocks depending on concentration of acid.)
3.Replace fluid with 5% sodium sulfate overnight
4.Wash 12 -24 hrs in running tap water.
5.Dehydrate in graded alcohols ,clear in chloroform or toluene and embed in wax

30. b)FORMIC ACID-SODIUM CITRATE
• Sodium citrate solution
Sodium citrate 100g
Distilled water 500ml
• Stock formic acid
Conc formic acid 250ml
distilled water 250ml

31. DECALCIFICATION
PROCEDURE
• Quantity of decal soln >20 vol. of
specimen.
• Wash the decalcified specimen
for 24-48 hrs –to remove the
decal soln.

32. Other Decalcifying Fluids
• Jenkins fluid
Absolute alcohol 73ml
Distilled Water 10ml
Chloroform 10ml
Glacial Acetic Acid 3ml
Hydrochloric Acid 4ml
• Trichloroacitic Acid –
Formal saline (10%) - 95 ml
Tricloroacitic acid - 5 gm
This is used for small biopsies. The process of decalcification is slow
hence cannot be used for dense bone or big bony pieces.

33. • Von –Ebners Fluid
Time Taken 3-5 Days
Formula:
Saturated Aq.Sodium Chloride 50ml
Distilled Water 50ml
Hydrochloric Acid 8ml

34. ION EXCHANGE RESINS
Used to
• remove the calcium ions from the fluid
• ensures a more rapid rate of solubility of the calcium from the tissue
• reduction in the time of decalcification.
Advantages :
1. Well preserved cellular detail
2. Faster decalcification
3. Elimination of the daily solution change
4. Resin can be reused by removing excess acid.

35. • Tissue is placed in a bottle in a
mixture of 10% or 20% resin
and formic acid.
• Resin used is ammonium form
of sulphonated polystyrene
resin.
• The volume of fluid is 20 – 30
times that of the specimen.
• After use, resin may be
regenerated by washing twice
with dilute N/10 HCl ,
followed by 3 washes in
distilled water.

36. ELECTROPHORETIC DECALCIFICATION
• First described in 1947.
• Attraction of the calcium ions to a negative electrode in
addition to the solution of the calcium in the electrolyte.
• Advantage
 Shortened time for complete decalcification.
 Better preservation of soft tissue details.
• Disadvantage
 Limited no. Of specimen processed at a time.

37. • A glass jar containing the acid decalcifying solution in which
is the electrode assembly and bone specimen, bone specimen
is suspended by a platinum wire anode in the jar.
• Used decalcifying fluid is
• 88% formic acid 100ml
• Hydrochloric acid 80ml
• Distilled water 820ml
• Current, causes an electric field between the electrodes,
enables the calcium ions to migrate rapidly from the specimen
(anode) to the carbon electrode (cathode).

39. • Temperature of the reaction- 30" to 45" C.
• Solutions changed after 8 hours of use to ensure maximum
speed of decalcification.
• Tissues are rinsed well in alkaline water & immersed in
lithium carbonate before staining.
• Lithium carbonate treatment of a cut section will neutralize
any remaining acid in the tissue

40. Histochemical techniques
Advantages
• It preserves the enzyme activity
• It also preserves the nucleic acids and polysaccharides.
It can be done by
Buffer mixture
Chelating agents

41. 1]Buffer mixtures
 Citric acid – citrate buffer (pH 4.5)
 Molar hydrochloric acid – citrate buffer (pH 4.5)
 Lorch’s citrate hydrochloric acid buffer (pH 4.5)
 Acetate buffer (pH 4.5)
• Calcium salts may be removed from bone when placed into a buffered
solution of citrate, pH 4.5.
• Daily changes of the buffer are necessary and the decalcification progress
checked by chemical oxalate test.

42. Chelating agents
Chelating agents are the organic compounds that have the power
of binding with certain metals.
• Advantages -
 It shows a minimum of artefact
 Section stained by most techniques with first class results.
• Disadvantages-
 slow process as calcium is removed layer by layer from the
hydroxyapatite lattice.

43. • First described by HILLMAN & LEE (1953)
• Commonly used agent is EDTA.
• Binds to metallic ions like Calcium & Magnesium
• Ionized calcium on the outside of the apatite crystal , the crystal
becomes progressively smaller during decalcification.
• Slow process that does not damage tissues or their stainability, also
pH sensitive.
• Excellent bone decalcifier for immuno histochemical or enzyme
staining & electron microscopy.

44. Surface decalcification
• Needed when partially decalcified bone/unsuspected mineral
deposits in soft tissue are found during paraffin sectioning.
• After finding a calcification, the exposed surface in a
paraffin block is placed face side down in 5% HCL for 1hour
or 10% formic acid for 15 to 60 minutes.
• Rinsed to remove the corrosive acids & re sectioned.

45. End point decalcification
• Probing the tissue with the needle
• Chemical tests
• Bubble test
• Radiography

46. • Physical tests require manipulation, bending probing or
trimming of the specimen to “feel” for remaining calcified
areas.
• Chemical test-(calcium oxalate test)
5 ml of decalcified fluid are neutralized with 0.5N sodium
hydroxide, 1 ml of 5 g/dl ammonium oxalate is added.
Appearance of turbidity indicates presence of calcium.
* Not done for EDTA Decalcification

47. • Bubble test
Acids react with calcium carbonate in bone to produce carbon dioxide , seen as
a layer of bubbles on the bone surface.
Bubble test is subjective & unreliable, tiny bubbles indicate less calcium
present.
• Radiography
Faxitron machine with exposure setting of 10-110 kv, 3ma tube current
And kodak x-omat x ray film is used.
Vinten Instruments Ltd,Jessamy Rd ,Weybridge England

48. Neutralization of acids
Chemical neutralization is accomplished by immersing decalcified
bone into either
• Saturated Lithium Carbonate Solution or
• 5-10% Aqueous Sodium Bicarbonate Solution for several hours.
• Many laboratories recommend rinsing the specimens in tap water
for a few hours.
• Culling(1974) recommended washing in two changes of 70%
alcohol for 12- 18 hour before continuing with dehydration

49. Processing decalcified bone
• Decalcified bone sectioning -made easier after infiltration and
embedding in harder paraffin to give firmer support.
• Small bone and needle biopsies containing little cortical bone can be
processed with soft tissues.
• Oversized, thick bone slabs require an extended processing schedule
to obtain adequate de-hydration, clearing and paraffin infiltration.
Ie., 3 changes of wax under vaccum of 2 hours

50. • If a bone sample still appears chalky, mushy and crumbles out of block during
sectioning, then:
 Dehydration, clearing or paraffin infiltration may be incomplete.
 Blocks can be melted down, and re- infiltrated with paraffin for up to eight
hours to see if this improves sectioning.
 Reversing processing by melting paraffin from bone and going back through
2 changes of xylene, 2 changes of 100% alcohol to remove residual water and
then reprocessing back in to paraffin.
 Double embedding procedure can produce better results than paraffin wax
alone.

51. • Base sledge microtome & wedge shaped steel or tungsten
carbide edged knife
• An optimal section thickness for bone is same as soft
tissues, 4-5µm or upto 6-7µm is accepted.
• Bone marrow biopsies should be cut at 2-3µm for marrow
cell identification
Microtomy

52. • The floating water bath may need to be hotter than for soft
tissues as bone has the tendency to crinkle when cut.
• Lifted onto the chrome-gelatin coated slides .

53. Staining methods for decalcified bone
• Hematoxylin and eosin: is still the primary stain used for
most final diagnoses with the aid of special stains.
Esp, Ehrlich’s & Gills .
• Collagen stains: Van Gieson picro – fuchsin, Masson’s
trichome.
• Silver –Reticulin Method , Picro-thionin stains.

54. UNDECALCIFIED SECTIONS
• In certain metabolic bone diseases like osteomalacia it is
valuable to assess the ratio of mineralized bone to non-
mineralized bone.
• 3methods to demonstrate osteoid seam :
* Adhesive tape method using Von Kossa
Technique
*Block Impregnation Method
*Resin Embedded sections

55. Adhesive tape method
Formal fixed ,paraffin embedded
Base sledge microtome
A strip of adhesive tape is pressed firmly against the Exposed
surface
Section bearing the tape is transferred to chrome-gelatin coated slide
Place another clean slide over the tape
Bulldog clip @
corners
In oven @56 degrees overnight
Remove the slide overing the tape & place wet filter paper for 30 mnts

56. Tape is removed by immersing the slide in warm xylene for 30-
60mnts
Von –kossa technique

57. Principle :
Calcium Phosphate + Silver Nitrate → Silver
Phosphate + Calcium Nitrate
Silver Phosphate → Metallic Silver (Light)
Von –kossa technique

58. Method
• Bring sections to water
• Rinse in distilled water
• Place in 5% silver nitrate in a glass coplin jar
• Wash well in distilled water
• Treat with 5% sodium thiosulphate for 5 mnts.
• Wash in running water for 3 mnts.
• Counter stain in Van-Gieson’s stain for 3 mnts.
• Dehydrate ,clear and mount in synthetic resin

59. Results
• Mineralized Bone – Black
• Osteoid Seams – Red

60. Iliac Biopsy – Severe Osteoporosis

61. 2)Block Impregnation Method
• In this method modified von kossa method is employed
• A Reducer solution is used –
Sodium Hypophosphite
0.1N Sodium Hydroxide
Distilled Water
RESULT:
Mineralized Bone –Black
Osteiod seams - Red

62. 3)Resin embedded sections
• Small blocks of undecalcified sections are processed into
methylmethacrylate
• Highly rigid microtome (JUNG K ) is used.
• Sections stored in 70% alcohol.
• Staining- H&E
Trichomes – Goldners modification of Masson’s T
Solochrome Cyanine stain
• Other methods - Fluorescent Labelling (Antibiotic tetracycline)
Microradiography

63. Teeth
• Teeth –same treatment as bone prior to sectioning.
Fixation : teeth should be fixed whole in NBF.
• Adult teeth will require up to 4 days of fixation
where as for younger teeth 24 hours of fixation
may be adequate.

64. • Decalcification: because of its high mineral concentration, enamel
is almost impossible to preserve through a completed decalcification
process.
• Brian (1966) used a long (approx.) 12 weeks of decalcification
procedure on a 3mm slice of tooth in a 4M sodium acetate – HCl
buffer solution at pH 3.55.
• Smith recommended 5 % trichloro acetic acid as a decalcifier,
while some prefer to decalcify teeth with EDTA or buffered formic
acid solutions.

65. • Radiography is ideal for progress and end point testing of
decalcification, reveals the presence of some metal
/amalgam fillings but not some implanted resin materials.
• Processing- Since tooth consists mainly of very dense
material, processing methods should be extended similar to
bone methods.

66. Microtomy
• A heavy microtome is used
• A sharp steel/tungsten carbide edged knife
• Thickness: 6-7μm
• Floating water bath temperature maintained just below
the melting point of the wax.
• Mounted on chrome-gelatin coated slide.
• Left in oven for 60mnts.

67. Staining
• Similar To Bone Stains
- Ehrlich’s H&E Over Meyer’s And Harris H&E
-Silver Reticulin Method
-Schmorl’s Picro-thionin

68. NOTE:
GOMORIS SILVER RETICULIN METHOD :
*To 4 parts of 10% aqueous silver nitrate add 1 part of 10%KOH .
*Allow the sample to settle down
*Remove the supernatant and wash the deposit twice with distilled
water.
*Add fresh,strong Ammonia drop by drop until the deposit is just
dissolved.
*Carefully add 10%silver nitrate drop by drop until the solution attains a
faint sheen.
RESULT :
Reticulin fibres- black
Collagen,cells,nuclei – Purple-grey

69. Schmorl’s Picro-Thionin Method
*Sections Are Washed Well
*Stain In Half Saturated Aqueous Thionin For 2-10mnts
*Wash Well In Running Water
*Stain In Saturated Aqueous Picric Acid For 30-60seconds
*Dehydrate, Clear And Mount
RESULT:
Dentinal tubules ,incremental lines ,lacunae and canaliculi- Dark
Brown
Cartilage ,Nuclei – Red/Brown
Background- Yellow

70. PROPRIETARY DECALCIFIER
• Components are trade secrets, product data sheet indicate if a
solution is rapid or slow.
• Rapid solution contain HCl , slow solution is a mixture of
buffered formic acid or formalin / formic acid.
• Usage is popular in busy laboratories , coz they are time &
cost effective & safe compared to strong acid.
• Disadvantage : formation of BCME ( BisChloroMethyl Ether)
,a potent carcinogen by the reaction between formaldehyde &
HCL.

72. Artefacts
Under decalcification
• Inability to section
• Incomplete infiltration of paraffin
• Staining characteristics
• Bone dust
• Remedy- surface decal, redecal

73. Over decalcification
• Nuclear detail lost or severely compromised
• Disruption of cell membrane and cytologic properties
• Loss of glycogen
• Swelling of tissue, especially collagen
• Staining characteristics
• Recalcification

74. Artefacts of overdecalcification

75. BoneDust

76. Recent studies
• Karpagaselvi Sanjai, Jayalakshmi Kumarswamy, and Lakshmi
Krishnan 2014
study was done to evaluate the rate of decalcification of six different
decalcifying agents and also their effect on staining characteristics on dental hard tissues.
namely,
neutral ethylene diamine tetra acetic acid (EDTA) decalcifying solution,
5% nitric acid, Perenyi's fluid,
formalin–nitric acid, 5% trichloracetic acid,
10% formic acid
Neutral EDTA was the most considerate to the soft and hard tissues and 5% nitric acid
was the least considerate to the tooth structure.

77. • Sung-Eun Choi, Soon Won Hong, and Sun Och Yoon(2015)
Bone marrow biopsy of 53 patients were decalcified according
to protocols of two comparison groups: EDTA versus HCl and
RDO GOLD (RDO) versus HCl for preserving cellular RNA,
DNA, and proteins and for molecular & immunohistochemical
analyses.
Result :
The EDTA protocol would be the best in preserving genetic
material. RDO may be an acceptable alternative when rapid
decalcification is necessary.

78. Recent advances
• Introduction of ultrasonic energization in
decalcification
Decalcification of bone specimens of 2-5 mm thickness can be
achieved in 5 hours or less when the decalcifying fluids are agitated by
ultrasonic energization.
Acid or chelating decalcifiers may be used and the application of
combined fixation-chelation permits routinely many histochemical
procedures previously requiring special handling.

79. • Decalcification by Perfusion
Using New Decal R -Hydrochloric acid 14%
PVP 7%
Aquadest 79%
trace surfactant.
The decalcifying agent was perfused at a rate of 6 ml/min and a
hydrostaticpressure of 120 cm for 30 min , 60 min, 90 min, 120
min and 240 min
• Result :
The tissues perfused 120 and 240 minutes, as well as those
immersed for 3 days in New DecalcR were all softened and easily
processed and viewed even under electron microscope.

80. • Microwave decalcification
Microwave-assisted decalcification saves from 10x to 100x of the
time required by routine methods.
The use of dilute acids (i.e. nitric or formic) in place of EDTA will
accelerate the process. The solution should be changed after each
cycle.
The temperature restriction between 42-45°C for best results

81. A decalcified section of cancellous bone (pink) and hyaline
cartilage (blue) from the epiphysis of a long bone (H&E). The
delicate trabeculae of the bone are well preserved as is the fine
structure of the bone marrow and associated adipocytes.

82. A decalcified section of compact bone from the shaft of a long bone
(H&E). The section is photographed under polarized light to demonstrate
the concentric lamelle forming the osteons. The birefringence is due to
the orientation of collagen fibres in the bone matrix which differs
between successive layers.

83. UNDECALCIFIED SECTIONS IN
OSTEOMALACIA

84. References
1. Bancroft JD, Marilyn Gamble. Theory and practice of histological
technique. 6th edition. Churchill Livingstone: Elsevier Health Sciences;
2008. pp. 53–105.
2. Jimson S ,Balachander N, Elumalai R “A Comparative Study in Bone
Decalcification Using Different Decalcifying Agents” 2319-7064 (2012)
3. Culling CFA, Allison RT, Barr WT. Cellular Pathology Technique. 4th
edition. Vol. 78. Butterworths (London, Boston): 1985. p. 611.p. 18.
4. Rolls OG, Farmer JN, Hall BJ. Artifacts in Histological and Cytological
Preparation. Scientia Leica Microsystems Education Series. April 2008;21

85. 5) Magnus Nilsson, Sten Hellstrom and Nils Albiin
“Decalcification by perfusion. A new method for rapid softening of
temporal bones”Histol Histopath (1991) 6: 415-420
6)Karpagaselvi Sanjai, Jayalakshmi Kumarswamy, Archana
Patil, Lokesh Papaiah, Srinivas Jayaram and Lakshmi Krishnan
“Evaluation and comparison of decalcification agents on the
human teeth” Oral Maxillofac Pathol. 2014 May-Aug; 16(2): 222–
227.
7) Sung-Eun Choi, Soon Won Hong, and Sun Och Yoon “Proposal
of an Appropriate Decalcification Method of Bone Marrow Biopsy
Specimens in the Era of Expanding Genetic Molecular Study” J
Pathol Transl Med. 2015 May; 49(3): 236–242

86. 8) Verdenius HHW and Alma L (1958) A quantitative study of decalcification
methods in histology J Clin Pathol. 1958 May; 11(3): 229–236
9) Mawhinney WH, Richardson E, and A J Malcolm (1984) Control of rapid
nitric acid decalcification. J Clin Pathol. 37(12): 1409–1413
10) Janneke C. Alers, Pieter-Jaap Krijtenburg, Kees J. Vissers, and Herman
van Dekken (1999)Effect of Bone Decalcification Procedures on DNA In Situ
Hybridization and Comparative Genomic Hybridization: EDTA Is Highly
Preferable to a Routinely Used Acid Decalcifier. Journal of Histochemistry and
Cytochemistry, Vol. 47, 703-710
11) P Sarsfield, C L Wickham, M V Joyner, S Ellard, D B Jones, and B S
Wilkins(2000) Formic acid decalcification of bone marrow trephines degrades
DNA: alternative use of EDTA allows the amplification and sequencing of
relatively long PCR products. Mol Pathol; 53(6): 336

87. 12) Yasuaki Shibata, et al (2000) Assessment of decalcifying protocols
for detection of specific RNA by non-radioactive in situ hybridization in
calcified tissues. Histochem Cell Biol 113:153–159
13) Yamamoto-Fukud T (2000) Effects of various decalcification
protocols on detection of DNA strand breaks by terminal dUTP nick end
labelling. Histochem J. 2000 Nov;32(11):697-702
14) Brown RSD, Edwards J, Bartlett JW, Jones C, and Dogan A (2002)
Routine Acid Decalcification of Bone Marrow Samples Can Preserve
DNA for FISH and CGH Studies in Metastatic Prostate Cancer. Journal
of Histochemistry and Cytochemistry, Vol. 50, 113-116
15) Recent Advances In Microwave Decalcification Protocols by
Herbert Skip Brown

89. Bullets …….
• Best decalcifying agent ??
• Why neutral EDTA is better ??
• Where is it commonly used??
• Disadvantage of EDTA
• Disadvantage of acid decalcification ?
• How do Bone and cementum, cartilage look after decalcification??
• Role of hydrofluoric acid??
• Depth of surface decalcifying fluid ??
• Can enamel be studied under decalcification?
• Partial decalcification ?? Where is it used ??
• Does enamel withstand decalcification ?
• % of inorganic content in enamel ?
• Why cant it be done in routine method ?
• What kind of blade is required ??