Alexa fluor 568 and 488 anti rabbit antibodies

Marrow stromal cells were flushed out from femur and tibia of WT and Fbn1^Prx1−/− mice. For LSK-HSC analyses, approximately 10⁶ bone marrow cells were labeled with antibodies anti-Lineage cocktail (APC), anti-CD34-FITC (clone RAM34), anti-Sca-1-PE-Cy7 (clone D7), anti-c-Kit-PE (clone 2B8) [27 (link)]. For CMP, MEP and GMP analyses, marrow cells were labeled with anti-FCγR-eFluor®450 (clone 93), anti-IL-7Rα-APC (clone A7R64), Lin-APC, anti-c-Kit-PE (clone 2B8), anti-CD34-FITC (clone RAM34) [28 (link)]. All antibodies were purchased from eBioscience (San Diego, Ca, USA). Samples were analyzed using a LSR II analyzer and FACSDIVA 6.1 software (BD Biosciences, San Jose, CA, USA); 3×10⁵ live cell events were recorded to analyze HSC, CMP, MEP and GMP frequency. For fibrillin-1 and macrophage visualization paraffin sections of tibiae from 3-month- old WT and Fbn1^Prx1−/− mice were incubated with anti-fibrillin-1 (a kind gift of Dr. L. Sakai) and anti F4/80 (clone BM8, Biolegend, San Diego, Ca, USA) whereas Alexa Fluor® 568 and 488 anti-rabbit antibodies (Molecular Probes, Eugene, OR, USA) were used for immunofluorescent labeling.

Fbn1-silenced Kusa-1 cells were grown in pro-adipogenic medium and analyzed by Oil-Red-O staining for intracellular fat droplets.^{(22 (link))} Total protein extracts from the same cells were analyzed by immunoblots using anti-PPARγ antibodies (Millipore, Billerica, MA, USA). Histomorphometric quantification of bone marrow fat content in the secondary spongiosa of proximal tibias were performed on two not-consecutive paraffin sections labeled with anti-perilipin antibodies (Cell Signaling Technology, Beverly, MA, USA) using Osteomeasure software. Anti-fibrillin-1 antibodies (a kind gift of Dr. L. Sakai) were employed to visualize Fbn1 expression in CFU-F cultures and bone cryosections;^{(15 (link))} goat anti-mouse LepR antibodies (R&D Systems, Minneapolis, MN, USA) were used to identify marrow MSCs in bone cryosections.^{(23 (link))} Alexa Fluor 568 and 488 anti-rabbit antibodies (Molecular Probes, Eugene, OR, USA) were used for immunofluorescent labeling. Anti-caspase-3 antibodies (Cell Signaling Technology, Danvers, MA, USA) were used to analyze protein extracts from primary bone marrow cultures.