Anti rabbit igg alexa594

In preparation for immunofluorescent staining, plated cardiomyocytes were washed once with PBS. The cells were fixed and permeabilized by applying Cytofix/Cytoperm für 20 minutes. After fixation, the cells were washed with Permwash (BD Biosciences) and stained with primary antibody dilutions for rabbit anti-cardiac troponin T (Abcam, 1:200) mouse anti-α-actinin (Abcam, 1:1000), rabbit anti-myosin light chain 2 (MLC2v) (Proteintech, 1:200), rabbit anti-connexin 43 (Abcam, 1:1000) and rabbit anti-N cadherin (Abcam, 1:200) overnight. The next day, the cells were washed and stained with the respective secondary antibodies anti-rabbit IgG Alexa594, goat anti-mouse IgG1 Alexa488, donkey anti-rabbit IgG Alexa488 or goat anti-mouse IgG1 Alexa594 (all Life Technologies, 1:1000). Next, the stained cardiomyocytes were washed, stained with DAPI to visualize nuclei and evaluated with a Zeiss Observer Z1 microscope.

For immunostaining, larval discs were dissected in PBS, fixed in 4% paraformaldehyde, blocked in 5% BSA, and incubated in primary antibodies overnight at 4°C, followed by secondary antibody incubation for 2 h at room temperature. The tissues were mounted with Vectashield mounting medium (Vectashield), and fluorescence images were acquired with a FluoView confocal microscope (Olympus). Primary antibodies used were rabbit anti-phospho-histone3 (Santa Cruz Biotechnology, Santa Cruz, CA, United States; 1:200), mouse anti-CD2 (AbD Serotec, 1:200), rabbit anti-CycE (Santa Cruz Biotechnology, 1:200), and goat anti-Diap1 (Santa Cruz Biotechnology, 1:200); and secondary antibodies were anti-mouse IgG Alexa 488, anti-rabbit IgG Alexa 594, and anti-goat IgG Alexa 594 (Life Technologies, 1:200).