Irdye 800 cw goat anti rabbit igg 926 32211

Urine analysis by immunoblot was performed according to Ferreira and colleagues [21 (link)]. Briefly, concentrated urine samples of each animal were diluted in Tris-buffered saline (TBS) to a final protein concentration of 0.1 µg·µL⁻¹. A volume of 100 µL was slot-blotted onto a nitrocellulose membrane (Hybond^® ECL™, Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK). Nonspecific binding was blocked with 5% (w/v) nonfat dry milk in TBS with Tween 20 (TBST). Each membrane was then incubated with the appropriate primary antibody solution [anti-matrix metalloproteinase (MMP)9 (ab38898) diluted 1:500 and anti-MMP2 (ab37150) diluted 1:200 from Abcam (Cambridge, UK)]. Membranes were then washed, incubated with the secondary antibody diluted 1:10,000 [IRDye^® 800 CW Goat anti-rabbit IgG (926-32211) from LI-COR Biosciences (Lincoln, NE, USA)] and washed again. All antibody solutions were diluted in 5% (w/v) nonfat dry milk in TBST and all incubations were performed for 1 h at room temperature with agitation. Detection was performed with fluorescence according to the manufacturers’ instructions (LI-COR Biosciences, Lincoln, NE, USA). Images were acquired using LI-COR Odyssey^® Scanner (LI-COR Biosciences, Lincoln, NE, USA) and analyzed using Image Studio™ Lite software (LI-COR Biosciences, Lincoln, NE, USA, v5.2.5). Optical densities (OD) values were expressed in arbitrary units.

Chloroquine (C129284) was purchased from Aladdin. Rapamycin (no. S1039) was obtained from Selleck Chemicals.
The antibody against LC3B (no. 2775) was purchased from Cell Signaling Technology. The antibodies against UAF1 (16503-1-AP), ATG7 (10088-2-AP), GAPDH (60004-1-lg), and SQSTM1 (18420-1-AP) were purchased from Proteintech. JEV E (GTX125867) was obtained from GeneTex. IRDye 800CW goat anti-rabbit IgG (926-32211) and IRDye 800CW goat anti-mouse IgG (926-32210) were purchased from LI-COR.