Procedure for in situ labeling of apoptotic cells in bone
Posted: Mon 16 Feb 2015 15:21Do you have a procedure for in situ labeling of apoptotic cells in bone and cartilage?
FAQ from Trevigen
A gentle method for the decalcification is required for bone and cartilage. Treatment of tissue will vary with size, porosity, and type of tissue. It is important to note that bone and cartilage can give high background in these assays due to poor quality or tissues. For example damage to samples can occur with incomplete fixation, roughened blade during sectioning, or strong proteinase K treatment.
Protocol Guideline:
1. Tissues should be fixed in 4% paraformaldehyde (10% neutral buffered formalin or 2-4% formaldehyde prepared in PBS) for 8 to 12 hours with one or two changes of paraformaldehyde solution. It may be necessary to gently separate the joint with dissection tools, while in fixative, to facilitate fixation throughout the entire tissue.
2. After fixation, the tissue is placed into 10 volumes of Decalcification Solution (recipe below) for up to 3 weeks at room temperature. It may be necessary to change the Decalcification Solution several times during this period. Decalcification should be allowed to continue until the tissue is pliable for sectioning. If further decalcification is required, it is possible to further decalcify sectioned tissues on slides.
3. Tissue should be placed into increasing ethanol series (70%, 95%, 100%) and then xylene prior to embedding in paraffin.
4. Sections should be cut at 5 µM, and floated onto glass slides treated for electrostatic adherence. Sections should be dried at 45˚C for up to one hour or at room temperature overnight. Slides are ready for labeling at this point.
Decalcification Solution:
To prepare two liters:
250 g EDTA, disodium salt
25 g sodium hydroxide
Make solution to 2 liters (~1750 mL deionized water)
pH should be approximately 7.0
FAQ from Trevigen
A gentle method for the decalcification is required for bone and cartilage. Treatment of tissue will vary with size, porosity, and type of tissue. It is important to note that bone and cartilage can give high background in these assays due to poor quality or tissues. For example damage to samples can occur with incomplete fixation, roughened blade during sectioning, or strong proteinase K treatment.
Protocol Guideline:
1. Tissues should be fixed in 4% paraformaldehyde (10% neutral buffered formalin or 2-4% formaldehyde prepared in PBS) for 8 to 12 hours with one or two changes of paraformaldehyde solution. It may be necessary to gently separate the joint with dissection tools, while in fixative, to facilitate fixation throughout the entire tissue.
2. After fixation, the tissue is placed into 10 volumes of Decalcification Solution (recipe below) for up to 3 weeks at room temperature. It may be necessary to change the Decalcification Solution several times during this period. Decalcification should be allowed to continue until the tissue is pliable for sectioning. If further decalcification is required, it is possible to further decalcify sectioned tissues on slides.
3. Tissue should be placed into increasing ethanol series (70%, 95%, 100%) and then xylene prior to embedding in paraffin.
4. Sections should be cut at 5 µM, and floated onto glass slides treated for electrostatic adherence. Sections should be dried at 45˚C for up to one hour or at room temperature overnight. Slides are ready for labeling at this point.
Decalcification Solution:
To prepare two liters:
250 g EDTA, disodium salt
25 g sodium hydroxide
Make solution to 2 liters (~1750 mL deionized water)
pH should be approximately 7.0