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Chondrocyte apoptosis after simulated intraarticular fracture: a comparison of histologic detection methods.

Dang AC, Kim HT - Clin. Orthop. Relat. Res. (2009)

Bottom Line: We observed differences between injured and uninjured areas of cartilage using the four methods.Human cartilage fixed in zinc-formalin and embedded in paraffin is amenable to programmed cell death analysis using any of four independent methods, each of which ostensibly has some advantages in terms of assaying different steps along the apoptotic pathway.Using the protocols described in this article, investigators may have additional tools to identify and quantify chondrocytes undergoing programmed cell death after experimental cartilage injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, University of California, 500 Parnassus Avenue, MU320 W, Box 0728, San Francisco, CA 94143, USA. alexis.dang@ucsf.edu

ABSTRACT
Accurate evaluation of programmed cell death, or apoptosis, in chondrocytes is essential to studying cartilage injury. We evaluated four methods of detecting chondrocyte-programmed cell death in formalin-fixed, paraffin-embedded cartilage after experimental osteochondral fracture. Human osteochondral explants were subjected to experimental fracture in a manner known to induce high levels of chondrocyte-programmed cell death. After 4 days in culture, specimens were fixed and analyzed for programmed cell death using: (1) terminal deoxynucleotidyl transferase end labeling; (2) DNA denaturation analysis using an antibody specific for single-stranded DNA; (3) immunohistochemistry using antisera specific for active caspase-3; and (4) in situ oligonucleotide ligation. Quantitative analysis of programmed cell death levels for each technique was performed comparing injured and uninjured areas of cartilage. We observed differences between injured and uninjured areas of cartilage using the four methods. Human cartilage fixed in zinc-formalin and embedded in paraffin is amenable to programmed cell death analysis using any of four independent methods, each of which ostensibly has some advantages in terms of assaying different steps along the apoptotic pathway. Using the protocols described in this article, investigators may have additional tools to identify and quantify chondrocytes undergoing programmed cell death after experimental cartilage injury.

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Photomicrographs (Stain, Safranin O (A) and DAB primary antibody staining with Fast Green counter stain (B–E); original magnification, ×10) compare (A) the injured zone and the control zone in (B) TUNEL, (C) ssDNA, (D) anti-active caspase-3, and (E) ISOL. The break in the cartilage is to the right of the injured zone images. TUNEL = terminal deoxynucleotidyl transferase end labeling; ssDNA = DNA denaturation analysis using anti single-stranded DNA antibody; ISOL = in situ oligonucleotide ligation.
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Fig1: Photomicrographs (Stain, Safranin O (A) and DAB primary antibody staining with Fast Green counter stain (B–E); original magnification, ×10) compare (A) the injured zone and the control zone in (B) TUNEL, (C) ssDNA, (D) anti-active caspase-3, and (E) ISOL. The break in the cartilage is to the right of the injured zone images. TUNEL = terminal deoxynucleotidyl transferase end labeling; ssDNA = DNA denaturation analysis using anti single-stranded DNA antibody; ISOL = in situ oligonucleotide ligation.

Mentions: All four methods when used for chondrocyte PCD showed differences when comparing the injury zone with the control (Table 1). In addition, similar spatial patterns of staining were observed using all techniques (Fig. 1). The magnitude of positive signal also was similar in all samples (Fig. 2).Table 1


Chondrocyte apoptosis after simulated intraarticular fracture: a comparison of histologic detection methods.

Dang AC, Kim HT - Clin. Orthop. Relat. Res. (2009)

Photomicrographs (Stain, Safranin O (A) and DAB primary antibody staining with Fast Green counter stain (B–E); original magnification, ×10) compare (A) the injured zone and the control zone in (B) TUNEL, (C) ssDNA, (D) anti-active caspase-3, and (E) ISOL. The break in the cartilage is to the right of the injured zone images. TUNEL = terminal deoxynucleotidyl transferase end labeling; ssDNA = DNA denaturation analysis using anti single-stranded DNA antibody; ISOL = in situ oligonucleotide ligation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2690763&req=5

Fig1: Photomicrographs (Stain, Safranin O (A) and DAB primary antibody staining with Fast Green counter stain (B–E); original magnification, ×10) compare (A) the injured zone and the control zone in (B) TUNEL, (C) ssDNA, (D) anti-active caspase-3, and (E) ISOL. The break in the cartilage is to the right of the injured zone images. TUNEL = terminal deoxynucleotidyl transferase end labeling; ssDNA = DNA denaturation analysis using anti single-stranded DNA antibody; ISOL = in situ oligonucleotide ligation.
Mentions: All four methods when used for chondrocyte PCD showed differences when comparing the injury zone with the control (Table 1). In addition, similar spatial patterns of staining were observed using all techniques (Fig. 1). The magnitude of positive signal also was similar in all samples (Fig. 2).Table 1

Bottom Line: We observed differences between injured and uninjured areas of cartilage using the four methods.Human cartilage fixed in zinc-formalin and embedded in paraffin is amenable to programmed cell death analysis using any of four independent methods, each of which ostensibly has some advantages in terms of assaying different steps along the apoptotic pathway.Using the protocols described in this article, investigators may have additional tools to identify and quantify chondrocytes undergoing programmed cell death after experimental cartilage injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, University of California, 500 Parnassus Avenue, MU320 W, Box 0728, San Francisco, CA 94143, USA. alexis.dang@ucsf.edu

ABSTRACT
Accurate evaluation of programmed cell death, or apoptosis, in chondrocytes is essential to studying cartilage injury. We evaluated four methods of detecting chondrocyte-programmed cell death in formalin-fixed, paraffin-embedded cartilage after experimental osteochondral fracture. Human osteochondral explants were subjected to experimental fracture in a manner known to induce high levels of chondrocyte-programmed cell death. After 4 days in culture, specimens were fixed and analyzed for programmed cell death using: (1) terminal deoxynucleotidyl transferase end labeling; (2) DNA denaturation analysis using an antibody specific for single-stranded DNA; (3) immunohistochemistry using antisera specific for active caspase-3; and (4) in situ oligonucleotide ligation. Quantitative analysis of programmed cell death levels for each technique was performed comparing injured and uninjured areas of cartilage. We observed differences between injured and uninjured areas of cartilage using the four methods. Human cartilage fixed in zinc-formalin and embedded in paraffin is amenable to programmed cell death analysis using any of four independent methods, each of which ostensibly has some advantages in terms of assaying different steps along the apoptotic pathway. Using the protocols described in this article, investigators may have additional tools to identify and quantify chondrocytes undergoing programmed cell death after experimental cartilage injury.

Show MeSH
Related in: MedlinePlus