-neutral ethylene diamine tetra acetic acid (EDTA) decalcifying solution

-5% nitric acid:nitric acid isalso known as aqua forties and sprit of niter is a
highly corrosive mineral acid
-Perenyi's fluid
-formalin–nitric acid
-5% trichloracetic acid
-10% formic acid:
- Potassium formate

Formic acid and formal-nitric acid were chosen since they are the most common weak
and strong demineralizing acids used respectively.
-sodium hydroxide
-Staind-dyes
haematoxylin and eosin:, especially useful for demonstrating the structure of
decalcified sections
Statistical Analysis: Paired sample t-test was done for inter-observer variation.
One-way-ANOVA and Post-hoc test was applied to compare the effects of different
decalcifying agents.
- The end point of decalcification was measured by physical method followed by
chemical and radiographic methods.
The physical method involved bending, needling and probing at the cervical area of
tooth using a fine needle or probe every day.
The chemical method employed was calcium – oxalate test, which involved the
detection of calcium by precipitation of insoluble Ca (OH)2 or calcium oxalate in
decalcifying solution.

Statistical Analysis: Paired sample t-test was done for inter-observer variation.
One-way-ANOVA and Post-hoc test was applied to compare the effects of different
decalcifying agents.

One way ANOVA was used for multiple group comparisons and Chi-square test was used
for analyzing categorical data. P value of 0.05/less was set for statistical
significance.

an ideal decalcifying agent should

Be fast;

Be good and;

Do good.

incisors, canines, premolars, and molarsnitric

DECALCIFICATION
1. DECALCIFICATION SHRAVYA.M
2. 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
3. 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.
4. Average thickness is 4-6 µm. To section the hard mineralised tissue
decalcification is necessary
5. 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.
6. 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.
7. 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
8. 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.
9. 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.
10. 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
11. 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.
12. 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.
13. Tissue damage during acid decalcification is four times greater when the tissue
is unfixed.
14. 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
15. Routine decalcification methods include: 1. Acids 2. Ion exchange resins 3.
Electrical ionization 4. Histochemical methods  Buffer mixtures  Chelating agents
5. Surface decalcification
16. 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
17. Strong acids • It Is An Inorganic Acid Eg: Nitric Acid, Hydrochloric Acid •
Recommended Concentration - 5-10% • They Decalcify Rapidly By Dissolving Calcium
18. MINERAL ACID DECALCIFIERS
19. 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
20. 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
21. 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
22. 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
23. 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.
24. WEAK ACID DECALCIFIERS ACETIC ACID PICRIC ACID
25. 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
26. • 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.
27. 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.
28. 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
29. b)FORMIC ACID-SODIUM CITRATE • Sodium citrate solution Sodium citrate 100g
Distilled water 500ml • Stock formic acid Conc formic acid 250ml distilled water
250ml
30. DECALCIFICATION PROCEDURE • Quantity of decal soln >20 vol. of specimen. • Wash
the decalcified specimen for 24-48 hrs –to remove the decal soln.
31. 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.
32. • Von –Ebners Fluid Time Taken 3-5 Days Formula: Saturated Aq.Sodium Chloride
50ml Distilled Water 50ml Hydrochloric Acid 8ml
33. 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.
34. • 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.
35. 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.
36. • 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).
37. • 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
38. 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
39. 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.
40. 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.
41. • 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.
42. 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.
43. End point decalcification • Probing the tissue with the needle • Chemical tests
• Bubble test • Radiography
44. • 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
45. • 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
46. 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
47. 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
48. • 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.
49. • 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
50. • 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 .
51. 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.
52. 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
53. 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
54. Tape is removed by immersing the slide in warm xylene for 30- 60mnts Von –kossa
technique
55. Principle : Calcium Phosphate + Silver Nitrate → Silver Phosphate + Calcium
Nitrate Silver Phosphate → Metallic Silver (Light) Von –kossa technique
56. 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
57. Results • Mineralized Bone – Black • Osteoid Seams – Red
58. Iliac Biopsy – Severe Osteoporosis
59. 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
60. 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
61. 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.
62. • 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.
63. • 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.
64. 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.
65. Staining • Similar To Bone Stains - Ehrlich’s H&E Over Meyer’s And Harris H&E -
Silver Reticulin Method -Schmorl’s Picro-thionin
66. 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
67. 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
68. 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.
69. Artefacts Under decalcification • Inability to section • Incomplete
infiltration of paraffin • Staining characteristics • Bone dust • Remedy- surface
decal, redecal
70. 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
71. Artefacts of overdecalcification
72. BoneDust
73. 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.
74. • 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.
75. 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.
76. • 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.
77. • 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
78. 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.
79. 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.
80. UNDECALCIFIED SECTIONS IN OSTEOMALACIA
81. 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
82. 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
83. 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
84. 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
85. 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 ??
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