Af647 goat anti rat igg

Immunostaining for Ki-67 (Vector), HER2 (AB16901; Abcam), and STAT1 (AB47425; Abcam) was performed as previously described^{6 (link)}. Secondary antibodies (AF488 AffiniPure donkey anti-mouse IgG, Cy3 AffiniPure donkey anti-rabbit IgG) were from Jackson ImmunoResearch Laboratories. Images were acquired with a Yokogawa spinning disk confocal on an inverted Nikon Ti fluorescence microscope using MetaMorph image acquisition software, and 3–5 fields were analyzed per tumor. Image analysis was performed using a semi-automated in-house platform (NIH ImageJ).
Immunofluorescence for FoxP3 (clone FJK-16s; eBioscience), CD8 (clone 4SM15; eBioscience), and Ki-67 (clone SP6; Thermo Scientific) was performed as previously described^{21 (link)}. Secondary antibodies (AF488 donkey anti-rabbit IgG, AF647 goat anti-rat IgG) were from Life Technologies. Tissues were counterstained with DAPI (Invitrogen). Images were acquired on a Nikon Eclipse Ni microscope using NIS Elements software, and 5–10 fields were analyzed per tumor.

Cardiomyocytes were fixed with pre-chilled methanol for 10 minutes, then washed twice using PBST (0.1% tween-20 in 1×PBS) with 5 minutes intervals. Cells were blocked for 1 hour at room temperature with blocking buffer (5% BSA in PBST) and incubated with primary antibodies overnight at 4°C. The primary antibodies were: Detyrosinated α-tubulin (Abcam, ab48389, used at 1:200), α-tubulin (CST, 3873S, used at 1:200), MARK4 (Abcam, ab124267, used at 1:200), APC anti-mouse CD45 (BioLegend, 103112, used at 1:200) and rabbit IgG isotype control (Novus Biologicals, NB810-56910, used at 1:2000). The cells were then washed with PBST and incubated with secondary antibody for 1 hour at room temperature. The secondary antibodies were: AF488 donkey anti-rabbit IgG (Invitrogen, A21206, used at 1:200), AF647 goat anti-mouse IgG (Invitrogen, A21236, used at 1:200), AF647 goat anti-rat IgG (Invitrogen, A21247, used at 1:200). DAPI (Sigma, 10236276001, used at 1:1000) was used. Confocal images were obtained by Leica SP5 Confocal Laser Scanning Microscope, collected by LAS AF software (2.7.3.9723), and analyzed by ImageJ (v2.0) analyze tools.