Freshly dissected long bones were fixed in a Formalin-based fixative at 4°C for 3 hours. Then the bones were embedded in 8% gelatin in PBS. Samples were snap frozen with liquid N2 and stored at −80°C. Bones were sectioned using a CryoJane system (Instrumedics). Sections were dried overnight at room temperature (RT) and stored at −80°C. Sections were re-hydrated in PBS for 5 min before immunostaining. 5% goat serum in PBS was used to block the sections. Primary antibodies were applied to the slides for 1h at RT followed by secondary antibody incubation for 30min at RT with repetitive washes in between. Slides were mounted with anti-fade prolong gold (Life Tech) and images were acquired on a Zeiss 710 confocal microscope. Rat-anti-CD41 (eBioscience, eBioMWReg30, Cat#13-0411, 1:100) was used as primary antibody.
Ebiomwreg30
Manufactured by Thermo Fisher Scientific
The EBioMWReg30 is a laboratory equipment product designed for molecular weight determination. It provides a reliable and accurate method for measuring the molecular weights of various biomolecules, such as proteins and nucleic acids, using electrophoresis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti fade prolong gold, by Thermo Fisher Scientific (2 mentions)
Facs lysing, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Gr 1 rb6 8c5, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd34 ram34, by Thermo Fisher Scientific (1 mentions)
Ter119, by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by BD (1 mentions)
4 protocols using ebiomwreg30
1
Immunostaining of Cryosectioned Bone
2
Immunostaining of Cryosectioned Bone
3
Quantitative Platelet Analysis via Flow Cytometry
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A small amount of peripheral blood (2 µL) was obtained from the tail vein. The blood was immediately transferred to a solution containing 47 μL of buffered HT solution (10 mM Hepes, 137 mM NaCl, 2.8 mM KCl, 1 mM MgCl 2, 12 mM NaHCO 3, 0.4 mM Na 2 HPO 4, bovine serum albumin (Citric acid 8 g/L, sodium citrate 22.4 g/L, glucose 2 g/L, sodium citrate; pH 5.1) and 0.5 μl of APC fluorophore anti-CD41a antibody (clone eBioMWReg30 eBioscience), followed by incubation for 30 min. After incubation, 250 μl of the lysis solution (FACS lysing-BD) was added, and incubated for another 10 min at room temperature, and then stored 4 °C. To perform platelet counts 40 µL of beads (SPHEROTM AccuCount Particles-Spherotech, Inc.) with known size and 40 µL of blood were used. The beads are larger than the platelets and do not present fluorescence, thus allowing the identification and quantification of the platelets. The acquisition was performed using the flow cytometer (FacsCalibur, BD), and analysed using the Cell Quest (BD) software.
4
Isolation of Murine Hematopoietic Stem Cells
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Bone marrow cells were obtained from the femora, tibiae, and iliac crests of 8- to 10-week-old female B6 or B6-Ly5.1 mice. c-Kit-positive cells were enriched by anti-c-Kit antibody-conjugated MACS beads (Miltenyi Biotechnology, Germany) and stained with a lineage cocktail consisting of allophycocyanin (APC)-eFluor 780-conjugated anti-B220 (RA3–6B2, eBioscience, Waltham, MA), Gr-1 (RB6-8C5, eBioscience), and Ter-119 (TER-119, eBioscience) antibodies. Cells were also stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD34 (RAM34, eBioscience), APC-conjugated anti-c-Kit (2B8, eBioscience), phycoerythrin-cyanine 7 (PE-Cy7)-conjugated anti-Sca-1 (D7, eBioscience), brilliant violet 421-conjugated anti-CD48 (HM48-1, eBioscience), PE-conjugated anti-CD150 (TC15-12F12.2, Biolegend), and PerCP-eFluor 710-conjugated anti-CD41 (eBioMWReg30, eBioscience) antibodies. CD150+CD41−CD48−CD34−c-Kit+Sca1+Lineage− (CD150+41−34−KSL) cells, which were designated HSC1, were sorted with a FACSAria III (BD Biosciences).
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