Alexa fluor 568 conjugated goat anti chicken igg antibody

Following a lethal injection of pentobarbital (intraperitoneal; ip), a transcardiac perfusion of phosphate buffered saline (PBS) followed by formalin was performed on a third cohort of mice (cohort 3, n ​= ​4/diet). Brains were placed in formalin for 6 ​h and then in a 30% sucrose solution for 24 ​h. Coronal sections of 25 ​μm were obtained using a microtome (Leica SM2000R). Staining against beta-galactosidase (βgal) was performed as previously described (Décarie-Spain et al., 2018 (link)). Briefly, sections were washed in PBS, blocked for 2 ​h in a 3% normal goat serum solution, permeabilized with Tween and incubated overnight with a polyclonal chicken βgal antibody (Ab9361, 1:1000; Abcam, Cambridge, UK) at 4 ​°C. The next day, sections were washed in PBS, incubated with an Alexa Fluor® 568-conjugated goat anti-chicken IgG antibody (Abcam, 1:1000), mounted on microscope slides with a DAPI mounting media (Vectashield, Vector Labs, Burlingham, CA, USA) and visualized on the fluorescent microscope (Zeiss AxioImager.M2 ApoTome.2). βgal-labeled cells within the NAc (core and shell combined) were quantified from coronal slices (anterior/posterior coordinates relative to bregma ranging from +1.42 ​mm to +0.845 ​mm; n ​= ​4/diet) using the “analyze particles” function of ImageJ (Schneider et al., 2012 (link)).

Heterozygous NFκB-LacZ mice subjected to the diet protocol (n = 4/group) were injected with a lethal dose of sodium pentobarbital prior to cardiac perfusion with 30 mL of ice-cold phosphate buffered saline (PBS), followed by 30 mL of 10% buffered formalin. Following 4 h immersion in 10% buffered formalin, brains were transferred to 30% sucrose for 24–48 h. Coronal brain sections (30 μm) containing the rostro-caudal extent of the NAc were sliced with a microtome (Leica SM2000R). Free floating slices were washed in PBS and incubated in a blocking buffer (3% normal goat serum + 0.1% Triton + 0.02% PBS-azide) for 2 h at room temperature. Cell membranes were then permeabilized by incubating sections in 0.2% Tween in PBS solution for 9 min and then rinsed with PBS prior to overnight incubation in blocking solution with a primary polyclonal chicken antibody against βgal, Ab9361 (1:1000), Abcam (Cambridge, UK) at 4 °C. Following washes, sections were incubated with an Alexa Fluor® 568 conjugated goat anti-chicken IgG antibody (Abcam, 1:1000) for 2 h, at room temperature. Following washes, sections were mounted onto microscope slides with DAPI aqueous mounting media (Vectashield, Vector Labs, Burlingham, CA, USA) and visualized (Zeiss AxioImager.M2 ApoTome.2).