Goat anti rabbit igg alexa 546

Cells were fixed with 4% paraformaldehyde at room temperature for 20 min and permeabilized with 0.5% Triton X-100 in PBS for 10 min. Then cells were incubated with primary antibodies overnight at 4 °C. Next, the cells were exposed to secondary antibodies conjugated to Alexa-488 Goat anti-Rabbit IgG (Invitrogen, A11008) Alexa-488 Donkey anti-mouse IgG (Invitrogen, A21202) or Goat anti-Mouse IgG Alexa-546 (Invitrogen, A21123), Goat anti-Rabbit IgG Alexa-546(Invitrogen, A11071) for one hour. Nuclear staining was performed using DAPI solution (Sigma, D9542).

Sectioning and immunostaining were done as described previously (Haubensak et al., 2010 (link)). Mice were perfused using 4% PFA. Brains were cryoprotected (15% sucrose) and frozen at −80°C until sectioning. Brains were sectioned on a cryostat (Leica, Biosystems) at either 30 μm thick (for direct mounting onto slides) or 60 μm thick (for free-floating sections collected in a staining disc). The following antibodies were used: rabbit anti-SF1 antibody (1:500, TransGenic), rabbit anti-SF1 antibody (1:500, Upstate), goat anti-c-Fos (Santa Cruz, 1: 500), rabbit anti-Esr1a (1: 500, Santa Cruz), mouse anti PR (1:500, Thermoscientific), rabbit anti-GFP (1:500, Invitrogen). The following secondary antibodies were used—donkey anti-goat IgG Alexa 546 (1:500, Invitrogen), goat anti-rabbit IgG Alexa 488 (1:500, Invitrogen), goat anti-rabbit IgG Alexa 546 (1:500, Invitrogen), goat anti-rabbit IgG Alexa 633 (1:500, Invitrogen). NeuroTrace fluorescent Nissl stains (1:200, Invitrogen) or DAPI (1:200, Invitrogen) was used to counterstain sections and label cell bodies. At least three representative coronal sections spaced equally along the AP axis were used for quantifications.

Collagen-II antibody: Samples were fixed for 25 min. The primary antibody, anti-collagen-II antibody (Abcam, Trumpington, UK), was used at a titre of 1:100 in ICC buffer and left refrigerated overnight. The secondary antibody, Alexa 546 goat anti-rabbit IgG (λ_Ex/λ_Em, 556/573 nm max, ThermoFisher Scientific, Waltham, MA, USA), at a titre of 1:100 in ICC buffer, was added at room temperature for 2 h. The samples were then washed and submerged in PBS for imaging.
DAPI and phalloidin: Once fixed and washed, phalloidin-TRITC (tetramethylrhodamine) was added at a titre of 1:1000 in PBS (λ_Ex/λ_Em, 540/565 nm max, ThermoFisher Scientific, USA) for 30 min at room temperature. The solution was removed, and the sample was washed twice with PBS. DAPI (4′,6-diamidino-2-phenylindole) was added at a titre of 1:1000 in PBS for 10 min (λ_Ex/λ_Em, 350/470 nm max, ThermoFisher Scientific, Waltham, MA, USA), and the sample was then washed and submerged in PBS for imaging.

Cells were fixed with 3.5% formaldehyde in the culture medium for 1 h at room temperature. After washing with SP buffer (0.8 M sorbitol, 25 mM potassium phosphate buffer, pH 7.5), cells were pretreated with 0.4 M β-mercaptoethanol for 30 min. After washing with SP buffer, cells were treated with Zymolyase 20T in SP buffer for 30 min to digest the cell wall. Immunofluorescence microscopy was performed according to a method described previously (Kilmartin and Adams, 1984 (link)). Primary antibodies used were anti-Ald4p antibody (rabbit polyclonal, 1:50 dilution, this study) and anti-porin (mouse, monoclonal, Molecular Probe Inc., 1:20 dilution). Alexa 546 goat anti-rabbit IgG (Molecular Probe) and Alexa Fluor 488 goat anti-mouse IgG (Molecular Probe) were used as secondary antibodies at 1:500 dilution. Immunostained cells were double-stained with DAPI (1 µg/ml). Preimmune serum was used in the same method as a control.

HeLa FlpIn cells were seeded into 24-well plates at a density of 75,000 cells per well on glass coverslips, HEK FlpIn cells at 100,000 cells per well on poly-L-lysin pretreated glass coverslips and expression of eGFP or eGFP-NLRP11 was induced with 1 µg/ml doxycycline. After overnight expression, cells were either infected with 160 hemagglutination units (HAU)/ml Sendai virus (SeV) for different durations, or directly fixed with 4% PFA in PBS, permeabilized with 0.5% Triton X-100 and blocked with 5% FBS in PBS. Cells were then incubated with primary and secondary antibody sequentially. Antibodies used: Primary: DDX3X (A300-474A Bethyl Laboratories), AIF (Cell Signalling # 4642). Secondary: Alexa-546 goat anti-rabbit IgG, Alexa-405 goat anti-mouse (Molecular Probes). DNA was stained with Hoechst 33258 (Sigma). Images were captured with a Leica DMi8 microscope using a HCX PL FL L 40X/0.60 or a HC PL APO 63X/1.40-0.60 OIL objective and processed using the Leica LasX software and ImageJ. For 3D deconvolution, Z-stacks of 4.05 µm depth were captured, with individual planes every 0.2 µm. Blind 3D-deconvolution was performed using the Leica LasX software, performing 10 iterations at a refractive index of 1.52. For quantitative analysis, sample pictures were blinded and counted by eye.

We used Rat anti-mouse LAMP1 (BD Bioscience, San Jose, CA), Rabbit anti-mouse α-Tubulin (Abcam), Rabbit anti-mouse γ-Tubulin (Abcam), Rabbit anti-mouse S4/19S RP (Abcam), Rabbit anti-mouse αβ/20S proteasome (Abcam), Rabbit anti-mouse α4/20S proteasome (Abcam), Mouse anti-mouse Ubiquitin P4D1 (Santa Cruz), Rabbit anti-mouse pSyk (Y525/526) (cellsignalling), Rabbit anti-mouse Syk (Abcam), anti-mouse β-actin (Abcam), Rat anti-mouse CD45R (BD Bioscience), Goat anti-mouse IgGFab² (Jackson ImmunoResearch), Goat anti-mouse IgM Fab² (Jackson ImmunoResearch). For secondary antibodies: Donkey anti-rabbit IgG-Alexa488 (LifeTech), Goat anti-rabbit IgG-Alexa546 (ThermoScientific), Donkey anti-rat IgG-Alexa546 (ThermoScientific), Donkey anti-rat-Alexa647 (ThermoScientific), Phalloidin-Alexa647 (ThermoScientific), DAPI (Abcam). Ovalbumin was purchased from Sigma-Aldrich, MG-132 and Epoxomicin were purchased from Merk (Millipore).