Alexa 488 secondary antibody

Aortic SMC were fixed at 24 hrs after plating by immersion in 2% paraformaldehyde in Dulbecco’s phosphate buffered saline (DPBS). Cells were then washed in a glycine buffer and incubated overnight at 4 °C with primary antibodies against integrin α2 (Abcam, San Francisco, CA, USA), integrin α5 (Milipore Sigma, Burlington, MA, USA), and smooth muscle α-actin (SMα-actin) (Millipore Sigma, Burlington, MA, USA) diluted in a sodium citrate buffer containing BSA and Triton X [24 (link)]. After washing, cells were incubated with Alexa 568 secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature, washed again and immediately imaged in DPBS. A similar procedure with overnight incubation was followed for the primary antibody against integrin β1 or β3 both pre-conjugated with Alexa 488 (BioLegends, San Diego, CA, USA). For smooth muscle γ-actin (SMγ-actin, Actg2) staining, cells were first fixed with 1% paraformaldehyde in DPBS followed by permeabilization with cold methanol [25 (link)]. Staining was performed as described by using an SMγ-actin primary antibody [26 (link),27 (link)] followed by Alexa 488 secondary antibody (Jackson Immuno Research, West Grove, PA, USA).

Glass coverslips containing the GT1-7 cells from each experimental group were rinsed with ice-cold PBS and fixed using 4% paraformaldehyde in PBS pH 7.4, at room temperature. After fixation, the cells were rinsed with PBS containing 0.1% Triton-X for permeabilization. After rinsing, the cells were incubated for 30 min in a blocking solution of 1% BSA in PBS containing 0.1% Triton-X. Cells were incubated overnight at 4 °C with the primary antibody (1:5000; rabbit anti-FoxO1, Cell signaling # 2880). After rinsing, the cells were incubated with donkey anti-rabbit IgG conjugated with Alexa 488 secondary antibody (1:500; Jackson Immuno Research: West Grove, PA, USA). Slides were then mounted using a medium containing DAPI (DNA staining, red). Photomicrographs were acquired using a Leica confocal laser scanning microscope. The immunoreactive structures were excited using argon or helium-neon green lasers with the excitation and barrier filters set for the fluorochrome used (green or red). Images showing the fluorescence were obtained from sequentially acquired images of slices excited by the laser.

Animals collected at the 6-day stage (precisely 144 hpf) were fixed in 4% PFA and 0.1% Triton X-100. Tubulin structures were marked with a monoclonal mouse antibody against alpha-acetylated tubulin (Cat# T6793, Sigma-Aldrich GmbH, Germany, 1 : 250 dilution) and an Alexa 488 secondary antibody (Jackson Laboratories, USA, 1 : 500 dilution). Nuclear DNA was stained with DAPI (1 : 1000 dilution) and membranes with mCLING–ATTO 647N (Synaptic Systems GmbH, Germany, 1 : 50 dilution). DAPI, tubulin and mCling fluorescence was excited at 405, 488 and 633 nm, respectively, and recorded with a Leica TCS-SP8 confocal microscope equipped with a 63× glycerol-immersion objective. For details, see chapter III in Chartier [34 ].

Sections were rinsed with 0.1 M PBS and incubated for 48 h at room temperature with rabbit anti-phospho AKT (1:4000, Cell signaling # 9275). After rinsing, sections were incubated with donkey anti-rabbit IgG conjugated with Alexa 488 secondary antibody (1:400; Jackson Immuno Research: West Grove, PA, USA). Finally, the sections were coverslipped with Fluoromont-G mounting medium (Southern Biotechnology Associates: Birmingham, AL, USA). Photomicrographs were acquired using a Leica confocal laser scanning microscope. The immunoreactive structures were excited using argon or helium-neon green lasers with the excitation and barrier filters set for the fluorochrome used (green). Images showing the fluorescence were obtained from sequentially acquired images of slices excited by the laser.

To analyze c-fos expression, coronal brain slices were incubated for 1 hr in blocking solution at room temperature (3% NGS, 0.3% Triton X-100) and then for 20 hr with anti-c-fos primary rabbit antibody (1:500, 226.003; Synaptic Systems, Göttingen, Germany) for 24 hr. Sections were washed and incubated with anti-rabbit Alexa-488 secondary antibody (1:500, Jackson Immunoresearch, West Grove, PA) for 2 hr at room temperature. Afterwards, sections were washed, mounted on slides and immediately coverslipped with Fluoromount-G with DAPI (Thermo Fischer). Images were examined by fluorescence microscopy (Leica DM 5000 B) and c-fos positive cells were counted bilaterally from two LC coronal slices for each animal with ImageJ.

Immunofluorescence assay was performed as described previously.¹⁹, ²⁰ In specific, brain slices were incubated with anti‐OxR2 (1:200, GeneTex) or orexin‐A antibody (1:200, MAB763, R&D Systems, USA) for 48 h, and then incubated with Alexa488 secondary antibody (1:1000, 711–545‐152, Jackson Immuno Research, USA) at room temperature for 2 h.