Alexa fluor 633 goat anti mouse igg h l

Mice were anesthetized with 125mg/kg Ketamine and 10mg/kg Xylazine cocktail intraperitoneal injection. The brain was fixed by the transcardial perfusion of 4% PFA/PBS, followed by overnight incubation in an additional 4% PFA/PBS. After removing the fixative, the brain was cryoprotected with a 10–30% sucrose solution. The section was made with 30μm slices. Blocking was achieved with 4% BSA in PBS and 0.3% Triton X-100. Primary antibodies and working dilution are the following: Mouse anti-β-Amyloid antibody (1:500, clone: 6E10, Biolegend), Rabbit anti-NeuN monoclonal antibody (clone: A60, Millipore), and Rabbit anti-Iba1 antibody (Wako). The tissue was then incubated with the following fluorescence-labeled secondary antibodies; Alexa Fluor 488 Goat anti-mouse IgG (H+L) (Invitrogen), Alexa Fluor 633 Goat anti-mouse IgG (H+L) (Invitrogen), Alexa Fluor 633 Goat anti-rabbit IgG (H+L) (Invitrogen). The core-dense amyloid plaques were stained with 0.5% Thioflavin S (Sigma, T1892). The nuclei were stained with Hoechst33342 dye. The images were taken on a Zeiss AiryScan 880 confocal microscope and further analyzed with ImageJ/Fiji^{50 (link)}. Five fields per section were randomly chosen in the cortical layer V and analyzed for quantification. The average value of 5 fields was presented for each mouse.

Mouse anti-NP monoclonal antibody (mAb) was prepared in our laboratory [30 (link)]. The following primary antibodies were obtained from commercial resources: rabbit anti-V5 polyclonal antibody (pAb) (AB3792, Merck Millipore, Billerica, MA, USA); rabbit anti-GAPDH pAb (10494-1-AP) and rabbit anti-HA tag pAb (51064-2-AP, Proteintech, Nanjing, China); mouse anti-Flag mAb (F3165) and rabbit anti-Flag pAb (F7425) from Sigma-Aldrich; and mouse anti-actin mAb (sc-47778) and mouse anti-SNX16 mAb (sc-271260, Santa Cruz, Dallas, TX, USA). The secondary antibodies used in Western blotting were Dylight 800 goat anti-rabbit IgG (RS23920) and Dylight 800 goat anti-mouse IgG (RS23910), purchased from Immunoway (Newark, DE, USA); the secondary antibodies used in confocal microscopy were Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A11034) and Alexa Fluor 633 goat anti-mouse IgG (H + L) (A21052), obtained from Invitrogen.

The mouse monoclonal antibodies (mAbs) against the PB2, PB1, PA, and NP proteins of IAV were generated in our laboratory [29 (link)]. The following primary antibodies were obtained from commercial sources: the rabbit anti-HACD3 polyclonal antibody (pAb) (28572-1-AP; Proteintech, Wuhan, China), rabbit anti-SQSTM1/p62 pAb (18420-1-AP; Proteintech), rabbit anti-Myc pAb (A00172; Genscript, Nanjing, China), mouse anti-Myc mAb (A00704; Genscript), rabbit anti-GST pAb (A00097; Genscript), rabbit anti-V5 pAb (AB3792; Sigma-Aldrich, Saint Louis, MO, USA), mouse anti-V5 mAb (V8012; Sigma-Aldrich), mouse anti-GAPDH mAb (60004-1-Ig; Proteintech), rabbit anti-HA pAb (11692-T62; Sino Biological, Beijing, China), rabbit anti-NA pAb (GTX125974; Genetex, Irvine, CA, USA), rabbit anti-M1 pAb (GTX125928; Genetex), rabbit anti-NS1 pAb (GTX125990; Genetex). The secondary antibodies, DyLight 800 goat anti-mouse IgG (H+L) (072-07-18-06; KPL, Gaithersburg, MD, USA) and DyLight 800 goat anti-rabbit IgG (H+L) (072-07-15-06; KPL), were used for Western blotting. The secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG (H+L) (A-11034; Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 633 goat anti-mouse IgG (H+L) (A-21052; Invitrogen), were used for confocal microscopy.

Immunofluorescent labeling of endogenous ASC was performed in fixed and permeabilized WT iBMDMs, as previously described [45 (link),47 (link)]. ASC specks were detected using purified anti-ASC (TMS-1, clone HASC-71) primary antibodies (1:500, Biolegend, San Diego, CA, USA) and Alexa Fluor 633 goat anti-mouse IgG (H+L) (1:200, Invitrogen, Thermo Scientific, Waltham, MA, USA) as secondary antibodies. A Leica TCS SP5 laser scanning microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Wetzlar, Germany) with the HCX plan apo 63× (numerical aperture 1.4) oil immersion objective was used for imaging. A 405 nm laser line of 20 mW diode laser was used for DAPI excitation with emission between 415 and 450 nm. A 1 mW 631-nm HeNe laser was used for Alexa Fluor 633 anti-ASC excitation with emitted light detected between 640–660 nm. LAS AF (Leica, Wetzlar, Germany) and ImageJ software were used to acquire and process images.

Snap frozen tissues from Ercc1^−/Δ and WT mice were lysed in RIPA lysis and extraction buffer (Thermo Fisher). Protein concentration was determined using BCA protein assay kit (Thermo Fisher). Equal amounts of protein were loaded onto SDS‐PAGE polyacrylamide gels then transferred to 0.2 µm pore size nitrocellulose membranes (Bio‐Rad). Membranes were blocked with 5% BSA in PBS‐Tween for 1 h at room temperature then incubated with primary antibodies overnight at 4℃. Membranes were then probed with secondary antibodies at room temperature. Protein expression was measured by fluorescence using iBright™ FL1000 Imaging System. The density of each blot was quantified by using ImageJ (NIH) normalized to GAPDH. The following primary antibodies were used in this study: rabbit anti‐GAPDH (Cell Signaling Technology, 2118), rabbit anti‐p16 (Santa Cruz, sc‐1207), rabbit anti‐p21 (Abcam, ab7960), rabbit anti‐p‐p65 (Cell Signaling Technology, 3033), mouse anti‐p‐p65 (Cell Signaling Technology, 6956), rabbit anti‐Pai‐1 (Santa Cruz, sc‐8979), rabbit anti‐COX2 (Cell Signaling Technology, 12282). The following secondary antibodies were used in this study: goat anti‐rabbit IgG (H+L) Alexa Fluor Plus 488 (Thermo Fisher, A32731), goat anti‐mouse IgG (H+L) Alexa Fluor 633 (Thermo Fisher, A21052).