Biochemistry: 2010

Tuesday, November 9, 2010

Von Kossa Stain for Calcium Detection

The method was originally developed by von Kossa, and underwent several modifications. The stain principle is a precipitation reaction in which silver ions react with phosphate (not calcium) in the presence of acidic material. Photochemical degradation of silver phosphate to silver then occurs under light illumination.

Additional methods need to be employed to confirm the presence of calcium and hydroxyapatite, such as Alizarin Red, which detects calcium deposits.

VON KOSSA Stain Procedure:

1. Sections to water.

2. Place sections in 1% silver nitrate solution under reasonable light for 30-60 minutes.

3. Wash in distilled water.

4. Treat with 3% sodium thiosulphate for 3-5 minutes.

5. Wash in water.

6. Counterstain with van Gieson for 3-5 minutes.

7. Wash in alcohol.

8. Clear .

9. Mount sections

RESULTS:

Mineralised bone black Osteoid red

Other Protols for Von Kossa

von Kossa Staining Protocol for Calcium

von Kossa Staining Protocol for Calcium. NovaUltra Special Stain Kits. Description: This technique is for demonstrating deposits of calcium or calcium salt ...
www.ihcworld.com/_protocols/special_stains/von_kossa.htm

Von Kossa Stain - Wikipedia, the free encyclopedia

Von Kossa Stain. From Wikipedia, the free encyclopedia. Jump to: navigation, search. Thevon Kossa stain is used to quantify mineralization in cell culture ...
en.wikipedia.org/wiki/Von_Kossa_Stain

Von Kossa's Method - Calcium
VON KOSSA'S METHOD - CALCIUM. PURPOSE: Abnormal deposits of calcium may be found in any area of the body. With the H&E stain, calcium appear deep ...
library.med.utah.edu/WebPath/.../VONKOSSA.PDF

Renal Fellow Network: the von Kossa Stain for Acute Phosphate ...

17 Aug 2009 ... A good detection method is the use of the von Kossa stain, which stains certain calcium-containing salts such as calcium phosphate a ...
renalfellow.blogspot.com/.../von-kossa-stain-for-acute-phosphate.html

Technical Bulletin #444 von Kossa Staining of Osteoclast ...
von Kossa Staining of Osteoclast Resorption on BD BioCoat™ Osteologic™ Discs ... Figure 1: von Kossa stain of BD BioCoat™ Osteologic™ Disc following ten-day ...
www.bdbiosciences.com/external_files/dl/doc/tech.../TB444.pdf

von Kossa staining alone is not sufficient to confirm that ...
Numerous techniques are currently used to characterize biological mineralization in intact tissues and cell cultures; the von Kossa staining method, ...
www.ncbi.nlm.nih.gov/pubmed/12724828

The von Kossa reaction for calcium deposits: silver lactate ...
We have demonstrated that positive von Kossa stain- ing correlates well with calcifications in... cultures the von Kossa stain usually yielded identifiable ...
www.springerlink.com/index/k782p815tn252t39.pdf

Re: [Histonet] von kossa stain

27 Jan 2004 ... My understanding of the von Kossa stain is that it is NOT specific for calcium. Rather, the silver nitrate in the staining solution (silver ...
www.histosearch.com/histonet/.../Re.Histonetvonkossastain.html

VON KOSSA STAIN KIT PROCEDURE
VON KOSSA STAIN KIT PROCEDURE. Item# KT 028. KIT. 100ml. Nuclear Fast Red Stain. 100ml. 5% Silver Nitrate. COMPONENTS. 100ml. 5% Sodium Thiosulfate ...
search.cosmobio.co.jp/cosmo_search_p/..._/KT028.20060119.pdf

von Kossa stain - Definition

von Kossa stain definition from the mondofacto online medical dictionary.
www.mondofacto.com/facts/dictionary?von+Kossa+stain

Von Kossa Staining Alone Is Not Sufficient to Confirm that ...
and cell cultures; the von Kossa staining method, elec- tron microscopic analysis (EM), ....However, the von Kossa stain is being used with greater ...
www.springerlink.com/index/TWUFXJY1VTHUVYR5.pdf

von Kossa and Alcian Blue Staining Protocol for Calcium and ...

Combination of von Kossa and Alcian Blue staining will make both calcium and mucosubstances visible on the same tissue sections. ...
www.ihcworld.com/_...stains/von_kossa.alcian_blue.htm

Histology Lab: VON KOSSA

Histotechnology Technical methods. Stain for calcium ... VON KOSSA. 1. Sections to water. 2. Place sections in 1% silver nitrate solution under ultra- ...
www.nottingham.ac.uk/pathology/protocols/vonkoss.html

Von kossa stain: Definition with Von kossa stain Pictures and Photos

Definition of Von kossa stain with photos and pictures, translations, sample usage, and additional links for more information.
www.lexic.us/definition-of/von_kossa_stain

Stain Expert™ - Your Stains & Reagents Information Center

Calcium Stain - Von Kossa. Celestine Blue Method - Lendrum. Copper Stain (Rubeanic Acid). Cresyl Violet Acetate Method for Helicobacter sp. ...
www.sigmaaldrich.com

JoVE: Undecalcified Bone Preparation for Histology ...
8 Jan 2010 ... An identical protocol or rack staining is necessary for ... Von Kossa. Place in silver nitrate solution and expose to strong light until ...
www.jove.com/index/details.stp?id=1707

Isolation of human multipotent mesenchymal stem cells from second ...
We developed a novel two‐stage culture protocol for isolating MSCs from amniotic .... (d) Osteogenic differentiation was demonstrated by von Kossa staining. ...
humrep.oxfordjournals.org/content/19/6/1450.full

BioMed Central | Full text | Isolation of mesenchymal stem cells ...
30 May 2007 ... Von Kossa staining of calcium-phosphate deposits was performed. ... (1999) [40] and a slightly modified protocol of the one reported by ...
www.biomedcentral.com/1472-6750/7/26

Fwd: [Histonet] Question on Von Kossa

Of course, as with any silver stain use acid cleaned glassware and gloves ... Does anyone have a good Von Kossa stain protocol that they would not mind ...
www.mail-archive.com/histonet@lists...edu/msg00672.html

Von Kossa Stain for Calcium Detection

Additional methods need to be employed to confirm the presence of calcium and hydroxyapatite, such as Alizarin Red, which detects calcium deposits.

VON KOSSA Stain Procedure:

1. Sections to water.

2. Place sections in 1% silver nitrate solution under reasonable light for 30-60 minutes.

3. Wash in distilled water.

4. Treat with 3% sodium thiosulphate for 3-5 minutes.

5. Wash in water.

6. Counterstain with van Gieson for 3-5 minutes.

7. Wash in alcohol.

8. Clear .

9. Mount sections

RESULTS:

Mineralised bone black Osteoid red

Other Protols for Von Kossa

von Kossa Staining Protocol for Calcium

Von Kossa Stain - Wikipedia, the free encyclopedia

Von Kossa Stain. From Wikipedia, the free encyclopedia. Jump to: navigation, search. Thevon Kossa stain is used to quantify mineralization in cell culture ...
en.wikipedia.org/wiki/Von_Kossa_Stain

Renal Fellow Network: the von Kossa Stain for Acute Phosphate ...

Re: [Histonet] von kossa stain

von Kossa stain - Definition

von Kossa stain definition from the mondofacto online medical dictionary.
www.mondofacto.com/facts/dictionary?von+Kossa+stain

von Kossa and Alcian Blue Staining Protocol for Calcium and ...

Combination of von Kossa and Alcian Blue staining will make both calcium and mucosubstances visible on the same tissue sections. ...
www.ihcworld.com/_...stains/von_kossa.alcian_blue.htm

Histology Lab: VON KOSSA

Von kossa stain: Definition with Von kossa stain Pictures and Photos

Definition of Von kossa stain with photos and pictures, translations, sample usage, and additional links for more information.
www.lexic.us/definition-of/von_kossa_stain

Stain Expert™ - Your Stains & Reagents Information Center

Calcium Stain - Von Kossa. Celestine Blue Method - Lendrum. Copper Stain (Rubeanic Acid). Cresyl Violet Acetate Method for Helicobacter sp. ...
www.sigmaaldrich.com

Fwd: [Histonet] Question on Von Kossa

Friday, January 15, 2010

Telomere and telomerase assay (2)

Relative Gene Expression in Normal and Tumor Tissue by Quantitative RT-PCR

Author(s): Dennis S. Salonga, Kathleen D. Danenberg, Jean Grem, Ji Min Park, Peter V. Danenberg

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:83; Page Range: 83-98

Abstract | Full Text | PDF (138K)

Quantitative Detection of Telomerase Components by Real-Time, Online RT-PCR Analysis with the LightCycler

Author(s): Thomas Emrich, Sheng-Yung Chang, Gerlinde Karl, Birgit Panzinger, Chris Santini

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:99; Page Range: 99-108

Abstract | Full Text | PDF (98K)

Standard TRAP Assay

Author(s): Angelika M. Burger

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:109; Page Range: 109-124

Abstract | Full Text | PDF (131K)

Stretch PCR Assay

Author(s): Jun-ichi Nakayama, Fuyuki Ishikawa

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:125; Page Range: 125-136

Abstract | Full Text | PDF (110K)

Fluorescent Detection of Telomerase Activity

Author(s): Wade K. Aldous, Amber J. Marean, Mary J. DeHart, Katherine H. Moore

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:137; Page Range: 137-146

Abstract | Full Text | PDF (99K)

Nonradioactive Detection of Telomerase Activity Using a PCR-ELISA-Based Telomeric Repeat Amplification Protocol

Author(s): Thomas Emrich, Gerlinde Karl

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:147; Page Range: 147-158

Abstract | Full Text | PDF (123K)

In Situ TRAP Assay Detection of Telomerase Activity in Cytological Preparations

Author(s): Kazuma Ohyashiki, Junko H. Ohyashiki

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:159; Page Range: 159-164

Abstract | Full Text | PDF (143K)

Biotinylated Primer for Detecting Telomerase Activity Without Amplification

Author(s): Daekyu Sun

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:165; Page Range: 165-171

Abstract | Full Text | PDF (81K)

Whole-Cell and Microcell Fusion for the Identification of Natural Regulators of Telomerase

Author(s): Henriette Gourdeau, Marsha D. Speevak, Lucie Jetté, Mario Chevrette

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:173; Page Range: 173-195

Abstract | Full Text | PDF (145K)

Screening with COMPARE Analysis forTelomerase Inhibitors

Author(s): Imad Naasani, Takao Yamori, Takashi Tsuruo

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:197; Page Range: 197-207

Abstract | Full Text | PDF (125K)

Telomerase as a Therapeutic Target: Therapeutic Potential of Telomerase Inhibitors

Author(s): John A. Double

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:209; Page Range: 209-216

Abstract | Full Text | PDF (71K)

Methods of Telomerase Inhibition
Additionally, methods are outlined in this chapter for determining the effectiveness oftelomerase inhibition through TRAP assays or assessment of telomere ...
www.ncbi.nlm.nih.gov/pmc/articles/PMC2423206/

Telomere and telomerase assay (1)

A telomere is a region of repetitive DNA at the end of a chromosome, which protects the end of the chromosome from deterioration. Its name is derived from the Greek nouns telos (τἐλος) "end" and merοs (μέρος, root: μερ-) "part".

Russian theorist Alexei Olovnikov was the first to recognize (1971) the problem of how chromosomes could replicate right to the tip, as such was impossible with replication in a 5' to 3' direction. To solve this and to accommodate Leonard Hayflick's idea of limited somatic cell division, Olovnikov suggested that DNA sequences would be lost in every replicative phase until they reached a critical level, at which point cell division would stop.

During cell division, the enzymes that duplicate the chromosome and its DNA cannot continue their duplication all the way to the end of the chromosome. If cells divided without telomeres, they would lose the ends of their chromosomes, and the necessary information they contain. (In 1972, James Watson named this phenomenon the "end replication problem".) The telomeres are disposable buffers blocking the ends of the chromosomes and are consumed during cell division and replenished by an enzyme, the telomerase reverse transcriptase.

Elizabeth Blackburn compared telomeres to the aglets (tips) on the ends of shoelaces that keep them from fraying.

In 1975–1977, Blackburn, working as a postdoctoral fellow at Yale University with Joseph Gall, discovered the unusual nature of telomeres, with their simple repeated DNA sequences composing chromosome ends. Their work was published in 1978. The telomere shortening mechanism normally limits cells to a fixed number of divisions, and animal studies suggest that this is responsible for aging on the cellular level and sets a limit on lifespans. Telomeres protect a cell's chromosomes from fusing with each other or rearranging—abnormalities which can lead to cancer—and so cells are normally destroyed when their telomeres are consumed. Most cancers are the result of "immortal" cells which have ways of evading this programmed destruction.

Elizabeth Blackburn, Carol Greider, and Jack Szostak were awarded the 2009 Nobel Prize in Physiology or Medicine for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase.

Telomeres and telomerase: biological and clinical importance ...
(15) developed an assay for testing telomerase activity in cell extracts. Based on identification of telomerase mechanisms and properties, the telomere ...
www.clinchem.org/cgi/content/full/43/5/708

Telomere Length Analysis and In Vitro Telomerase Assay.

1 Introduction; 2 Materials and Methods; 2.1 Telomere Measurement; 2.2 Telomere Cloning; 2.3 TRAP Telomerase Assay; 3 Notes; References. Browse by Subject ...
www.springerprotocols.com/Abstract/doi/10.../1-59259-434-4:123

Facts about Telomeres and Telomerase

The following section will teach you the basics of telomeres and telomerase. It will also introduce you to the potential applications of current telomerase ...
www4.utsouthwestern.edu/cellbio/shay.../sw_facts.html

TRAP–LIG, a modified telomere repeat amplification protocol assay to quantitate telomerase inhibition by small molecules.
Its major function is to maintain the length of telomeric DNA by synthesizing telomeric DNA repeats, and its enzymatic activity is assayed by means of the ...
linkinghub.elsevier.com/retrieve/pii/S0003269708002960

Telomeres and Telomerase: The Cellular Timekeepers

The telomeres appear to be the cellular timekeepers, but are they related to human aging?
www.senescence.info/telomeres.html

Effect of Telomere and Telomerase Interactive Agents on Human Tumor and Normal Cell Lines.
The cytotoxic effect of telomere and telomerase interactive agents against normal human marrow cells in the colony-forming assay showed the same range of ...
clincancerres.aacrjournals.org/content/6/3/987.full

Cell Research - Telomere and telomerase in oncology
Shortening of the telomeric DNA at the chromosome ends is presumed to limit the lifespan of... Telomere, telomerase, cancer, telomerase assay, inhibitor ...
www.nature.com

Telomeres and telomerase — Genes & Development
Cdc13p (gray) and Est1 (purple) may recruit telomerase to the telomeric 3′ end in ..... (1995) An in vitro assay for Saccharomyces telomerase requires EST1. ...
genesdev.cshlp.org/content/13/18/2353.full

Telomerase Assay in Renal Cancer

Bacchetti, S. and Counter, C. M. (1995) Telomeres and telomerase in human cancer. ...quantitative nonisotopic assay for telomerase activity in humantumors. ...
www.springerprotocols.com/Abstract/doi/10.1385/1-59259-144-2:05

Introduction to Telomeres and Telomerase

Author(s): Michael C. Bibby

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:01; Page Range: 1-12

Abstract | Full Text | PDF (81K)

Detection of Chromosome Ends by Telomere FISH

Author(s): Harry Scherthan

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:13; Page Range: 13-31

Abstract | Full Text | PDF (117K)

Telomere Length Distribution: Digital Image Processing and Statistical Analysis

Author(s): Jean-Patrick Pommier, Laure Sabatier

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:33; Page Range: 33-63

Abstract | Full Text | PDF (351K)

Analysis of Telomerase RNA Gene Expression by In Situ Hybridization

Author(s): W. Nicol Keith, Joseph Sarvesvaran, Martin Downey

Pub. Date: Mar-01-2002; DOI:10.1385/1-59259-189-2:65; Page Range: 65-81

Abstract | Full Text | PDF (132K)

Protein Purification Protocols (2)

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Protein Purification (from Wikipedia, the Free Encyclopedia)

Contents

1 Purpose

2 Strategies

3 Evaluating purification yield

4 Purification of a tagged protein

5 Methods of protein purification

6 Extraction

7 Precipitation and differential solubilization

8 Ultracentrifugation

9 Chromatographic methods

9.1 Size exclusion chromatography