Whole-mount tissue preparation of sternal bones was performed as described (Kunisaki et al., 2013 (link)). In brief, sternal bones from tamoxifen-induced Pdzk1ip1-CreER R26SW were isolated, transected into two to three fragments and sagittally bisected to expose the BM. The tissues were fixed in 4% paraformaldehyde for 1 h at 4°C, stained with biotinylated anti-CD48 (HM48-1; BioLegend), rat anti-mouse CD41 (MWReg30; BioLegend), and biotinylated lineage markers (anti-Ter119 [TER119], anti-B220 [6B2], anti-Gr1 [RB6-8C5], anti-CD3e [500A2], anti-CD11b [HM1/70]; eBiosciences). Secondary reagents included Alexa Fluor 647–conjugated goat anti-rat IgG and Alexa Fluor 488–streptavidin conjugate (Life Technologies). Tissues were imaged on a 35-mm glass-bottom dish using a Zeiss LSM 710 confocal microscope.
Protocol detail
Sternal Bone Whole-Mount Imaging
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HM48-1;
(MWReg30;
Alexa Fluor 647–conjugated goat anti-rat IgG
Alexa Fluor 488–streptavidin conjugate
LSM 710 confocal microscope
Alexa fluor 488
Alexa fluor 647
Anti igg
Bones
Bones tissue
Cd11b
Confocal microscope
Dish
Goat
Mouse
Paraformaldehyde
Sternal
Streptavidin
Tamoxifen
Tissues
Corresponding Organization : New York University
Other organizations : Columbia University Irving Medical Center, New York Genome Center
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Variable analysis
independent variables
- Tamoxifen-induced Pdzk1ip1-CreER R26SW
- Sternal bone morphology and cell composition
- Sternal bone fragments
- Fixation in 4% paraformaldehyde for 1 h at 4°C
- Staining with biotinylated anti-CD48, rat anti-mouse CD41, and biotinylated lineage markers
- Secondary reagents including Alexa Fluor 647–conjugated goat anti-rat IgG and Alexa Fluor 488–streptavidin conjugate
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