Dylight conjugated secondary antibody

Cell cultures were monitored daily using phase contrast microscopy. After 7 days of culture, RNA samples were collected using an RNA collection kit following manufacturer protocol (Applied Biosystems, Carlsbad, CA, USA). mRNA samples were then reverse transcribed to DNA using a cDNA kit following manufacture protocol (Applied Biosystems). The resulting DNA samples were analyzed with quantitative real-time polymerase chain reaction (qRt-PCR) following manufacturer protocol (Applied Biosystems). mRNA sequences of interest included oct 4, sox2, connexin 43, GATA4, cardiac troponin T (cTnT), ryanodine receptor 2 (RYR2), L-type calcium channel subunit alpha-1c, and GAPDH (Applied Biosystems).
After 7 to 14 days of culture, cell cultures were also fixed with paraformaldehyde (Alfa Aesar, Ward Hill, MA, USA) at 4°C for 20 minutes. Fixed cells were incubated with antibodies against GATA4 (rabbit polyclonal, Sigma-Aldrich) and cardiac troponin T (mouse polyclonal; Abcam, Cambridge, MA, USA), then in DyLight-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and DAPI with VectaShield (Vector, Burlingame, CA, USA). The cell were imaged using an epifluorescence microscope (DMI 6000B, Lieca Microsystems, Bannockburn, IL, USA).

Immunocytochemistry of cultured neurons was performed as previously described (An et al., 2008 (link)). The following primary antibodies were used: rabbit anti-GFP (Clontech Laboratories), 1:5,000 dilution; mouse anti-GFP (BD Bioscience), 1:1000 dilution; mouse anti-Myc (Sigma Aldrich), 1:5000 dilution; rabbit anti-Myc (Cell Signaling Technology) 1:1,000 dilution; mouse anti-Flag (Sigma Aldrich); mouse anti-p75 (R&D Systems) 1:1,000 dilution. For protein distribution, fluorescent immunocytochemistry was performed. Appropriate DyLight-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove) and used at a dilution of 1:500. For spine density and morphology analyses, immunocytochemistry was performed using a biotinylated secondary antibody and an avidin-biotin complex solution (Vector Labs). Immunoreactivity was visualized using a solution containing 0.05% DAB and 0.003% H₂O₂ in 100 mM Tris pH7.5. After colorimetric reaction was complete, cells were rinsed with 100 mM Tris and dehydrated, then coverslips were mounted onto glass slides using DPX mounting medium.

Immunolocalization of Ki67, CD31, ESR1, PGR, p-IKK, p-STAT3, CD68, CD163, COX2, iNOS, MMP9 and p-AKT was determined in cross-sections (5 μm) of paraffin-embedded uterine sections using specific primary antibodies and a Vectastain Elite ABC Kit (Vector laboratories, Burlingame, CA, USA) or DyLight-conjugated secondary antibody (Jackson ImmunoResearch Lab, West Grove, PA, USA). Antibodies used in these analyses were: anti-CDH1 (1:120 dilution, 610181, BD Biosciences, San Jose, CA, USA), anti-Ki67 (1:200 dilution, 550609, BD Biosciences), anti-CD31 (1:100 dilution, ab28364, Abcam, Cambridge, MA, USA), anti-ESR1 (1:100 dilution, sc-542, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PGR (1:200 dilution, RB-9017-P0, Thermo Scientific, Rockford, IL, USA), p-IKK (1:150 dilution, 2697, Cell Signaling Technology, Danvers, MA, USA), p-STAT3 (1:50 dilution, 9145, Cell Signaling Technology), CD68 (1:300 dilution, ab955, Abcam), CD163 (1:300 dilution, ab126756, Abcam), COX2 (1:50 dilution, RM-9121, Thermo Scientific), iNOS (1:50 dilution, 610333, BD Biosciences), MMP9 (1:50 dilution, 3852, Cell Signaling Technology), and p-AKT (1:200 dilution, 3787, Cell Signaling Technology).