Rat anti mouse itga5 biotin

Mouse embryos were fixed at 4°C with Stefanini solution; 20 g/L Paraformaldehyde, 6.667% saturated picric acid and equal amounts of Sörensen Buffer 0.2M, pH7.2 and distilled water, followed by equilibration in 20% sucrose prior to 10 μm cryo-sectioning. Sections were permeabilized in PBS/0.1% Triton X-100, blocked with 10% goat or donkey serum (Sigma-Aldrich) and stained with primary antibodies: chicken anti-mouse eGFP (Millipore), rabbit anti-mouse MYL2 (Synaptic Systems), rat anti-mouse ITGA6 Allophycocyanin (APC) (eBioscience), rat anti-mouse ITGA5 Biotin (LifeSpan Biosciences) and rabbit anti-mouse cTropT PE (BD Biosciences). Primary antibodies were visualized with secondary antibodies conjugated to FITC/Cy3/Cy5 (Jackson ImmunoResearch) or Alexa Fluor 555/647 (Invitrogen). Hoechst 33342 (Invitrogen) was used to localize nuclei. Imaging was performed using a Zeiss LSM 780 (Germany) laser scanning confocal microscope or a Leica DM500 B (Switzerland).

Embryonic nkx2.5-eGFP heterozygous and wild-type heart cell suspensions were stained in PBS containing 5% FCS with the following antibodies: rat anti-mouse Flk-1 Pacific Blue (BioLegend), rat anti-mouse CD49f (ITGA6) Allophycocyanin (APC) (eBioscience), hamster anti-mouse CD49a (ITGA1) PE (BD Biosciences), rat anti-mouse ITGA5, Biotin (LifeSpan Biosciences), sheep anti-mouse CDH2 (R&D) and/or rabbit anti-mouse cTropT PE (BD Biosciences). Streptavidin Qdot605 (Invitrogen) and donkey anti-sheep FITC (R&D) were used for secondary detection. To exclude non-viable cells, cells were stained with Fixable Viability Dye eFluor 780 (eBioscience). For intracellular staining, the cells were fixed in 4% PFA for 15 minutes, following by permeabilization in 0.1% Triton X-100 (3 minutes) and blocking in 1% BSA for 30 minutes. The cytometric acquisition and sorting was performed on Aria III (BD Biosciences) or Influx (BD Biosciences) using the 100 μM nozzle. Positive gates were set according to unstained, FMO and single stain controls. Small aliquots of cells were re-analyzed using the same settings for gating to assess purity.