Alexa 647 conjugated goat anti rabbit igg

AAV2/9.hSynapsin.tdTomato.T2A.mGIRK2-1-A22A.WPRE-injected mice were deeply anesthetized with an i.p. injection of avertin (250 mg/kg). Mice were transcardially perfused with ice-cold phosphate-buffered saline (PBS) containing (in mM) 137 NaCl, 1.5 KH₂PO₄, 8 NaH₂PO₄, 2.7 KCl (pH = 7.4), followed by 4% paraformaldehyde in PBS. The brains were fixed overnight in 4% paraformaldehyde. Fixed brains were sectioned coronally in 25 μm sections. Free-floating sections were permeabilized with 0.2% Triton X-100 in PBS (PBS-T) and blocked in 5% normal goat serum in PBS-T for one hour at room temperature. Slices were washed and incubated with 1:500 polyclonal rabbit anti-Kir3.2 antibodies (Alomone) in PBS overnight at 4°C. Sections were incubated with 1:500 Alexa 647-conjugated goat-anti-rabbit IgG (Invitrogen) in PBS for 2 hours at room temperature. Slices were mounted onto microscope slides (Fisher Scientific). Fluorescent confocal images were obtained using a Zeiss LSM 510 META laser scanning confocal microscope (Carl Zeiss) with a 40x Plan-Neofluar, NA 1.3, oil-immersion lens. All images were processed using ImageJ.

The excised tumors and organs were cryosectioned at 10 μm, fixed
with 4% of paraformaldehyde in PBS, and immunostained with rabbit
anti-fluorescein (Life Technologies, USA) and rat anti-mouse CD31 (BD
Biosciences, USA) as primary antibodies, and with Alexa 488-conjugated goat
anti-rabbit IgG and Alexa 647-conjugated goat anti-rat IgG (Invitrogen, USA) as
secondary antibodies. Alternatively, the tissue sections were immunostained with
rabbit anti-NRP-1 (Abcam, UK) or rabbit anti-αv-integrin (Millipore, US)
followed by Alexa 647-conjugated goat anti-rabbit IgG (Invitrogen, USA). The
nuclei of cells were stained with 10 μg/ml DAPI. Confocal images of the
tissue sections were analyzed with the ZEN lite 2012 image software.