Alexa fluor 488 conjugated goat anti mouse igg h l

To clarify the cellular localization of the wild-type and mutant 3a proteins of SARS-CoV, HeLa cells were cultured on coverslips and transfected with 1 μg of pCA7-flag-3a or pCD7-flag-3a-CS together with 0.5 μg of ER-mCherry or DsRed-Golgi (Ito et al., 2012 (link)). At 24 h post-transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100/PBS. After washing with PBS and blocking with 4% BSA/PBS, the cells were incubated with a mouse anti-flag antibody (M2, Sigma) followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (Life Technologies).
To observe the cellular distribution of NLRP3 in the E- or 3a-expressing cells, HeLa cells were cultured on coverslips and transfected with 1 μg of pCA7-HA-E, pCA7-HA-EV25F, pCA7-HA-3a, pCA7-HA-3a-CS, or pCA7 control vector together with 0.5 μg of pCA7-NLRP3. At 24 h post-transfection, cells were fixed and permeabilized with 4% paraformaldehyde and 1% Triton X-100/PBS. After washing and blocking, the cells were incubated with rabbit anti-HA (561, MBL) and mouse anti-NLRP3 (Cryo-2; AdipoGen) antibodies, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) and Alexa Fluor 568-conjugated goat anti-mouse IgG (H+L) (Life Technologies). Fluorescent signals were observed by confocal microscopy (A1R⁺, Nikon).

Antigen retrieval was performed using 0.01 M citrate buffer (pH 6.0) at 95°C for 20 min. The sections were processed for 0.25% Triton X‐100 treatment for 15 min, blocked with 5% goat serum in PBS for 1 h at room temperature (RT) and then incubated with corresponding primary antibodies (prepared in goat serum): anit‐RBM24 (Abcam, Cat no: ab94567, 1:300), anti‐Ki‐67 (Abcam, Cat no: ab15580, 1:300), anti‐pHH3 (Abcam, Cat no: ab5176, 1:300), anti‐PTEN (Santa Cruz, Cat no: SC‐7974, 1:300) overnight at 4°C. After washing five times with PBS, the sections were incubated with the appropriate secondary antibodies (prepared with goat serum) AlexaFluor^®488‐conjugated goat anti‐rabbit IgG (H+L) (Life Technologies, Cat no: A‐11008, 1:500), AlexaFluor^®488‐conjugated goat anti‐mouse IgG (H+L) (Life Technologies, Cat no: A‐11001, 1:500), or AlexaFluor^®555‐conjugated goat anti‐rabbit IgG (H+L) (Life Technologies, Cat no: A‐21428, 1:500) at RT in the dark for 1 h following washes with PBS. Next, slides were treated with the fluorescent blue dye Hoechst 33258 (Sigma, 1:1000) at RT for 10 min. After washing with PBS again, the slides were sealed with 50% glycerin. Representative pictures were photographed using a fluorescence microscope (Olympus, Japan).

HEK293T cells and PAMs, which were stored in our laboratory, were cultured in RPMI 1640 medium (catalog no. C11875500BT; Gibco) supplemented with 5% antibiotics-antimycotics (10,000 IU/mL penicillin, 10,000 μg/mL streptomycin, and 25 μg/mL amphotericin B) (catalog no. 15240-062; Gibco) and 10% heat-inactivated fetal bovine serum (FBS) (catalog no. 10099-141C; Gibco) in a 37°C incubator in 5% CO₂. The ASFV pig/Heilongjiang/2018 (ASFV HLJ/2018) strain was isolated from field samples from China as described previously (63 (link)). ASFV-ΔH240R was generated in our previous study (8 (link)).
A rabbit anti-Myc PAb (catalog no. ab9106; Sigma-Aldrich), a mouse anti-Flag MAb (catalog no. ab62928; Sigma-Aldrich), and a rabbit anti-Flag PAb (catalog no. ab1162; Sigma-Aldrich) were purchased from Sigma-Aldrich. Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) (catalog no. 2072687; Invitrogen), Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (catalog no. 1942237; Invitrogen), Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) (catalog no. 111-585-003; Invitrogen) and Alexa Fluor 568-conjugated goat anti-mouse IgG (H+L) (catalog no. 175697; Invitrogen) antibodies were purchased from Invitrogen. A mouse anti-GST MAb (catalog no. K200006M; Solarbio) and 4′,6-diamidino-2-phenylindole (DAPI) (catalog no. C006; Solarbio) were purchased from Solarbio.

The presence of aquaporin-5 and ZO-1 in 3D cultured cells was confirmed by indirect immunofluorescence staining as described previously [33 (link)]. Briefly, samples were fixed with 10% formaldehyde/PBS for 10 min and permeabilized by PBS containing 1% BSA/0.2% Triton X-100 for 15 min at room temperature. The samples were incubated with primary antibodies against aquaporin-5 and ZO-1 (1:50 dilution) overnight at 4°C. After three washes with PBS/0.05% Tween 20, the samples were incubated with a secondary antibody (Alexa Fluor 488-conjugated goat anti-mouse IgG [H+L]; Invitrogen, CA), diluted 1:500 for 2 h at room temperature. The cell nuclei were counterstained by propidium iodide for 5 min and subsequently washed three times with PBS/0.05% Tween 20. The stained cells were visualized using a Nikon A1⁺ confocal microscope (Japan). The average intensity value of the nonspecifically bound secondary antibody was subtracted from all images using the ImageJ program.

CAPE (purity ≥98%) was obtained from Nature Standard (Shanghai, China). Concanavalin A type IV (ConA), lipopolysaccharide (LPS), Percoll, Triton X-100, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Recombinant mouse IL-12 (rIL-12; p70); purified Ultra-LEAF™ anti-mouse IL-4 antibody; purifiedLEAF™ anti-mouse CD3 antibody; purified LEAF™anti-mouse CD28 antibody; antibodies against mouse CD4, IL-17A, IFN-γ, Foxp3, CD25, and CD69; cell activation cocktail; brefeldin A solution; fixation/intracellular staining permeabilization wash buffer; and Foxp3 fixation/permeabilization buffer were obtained from BioLegend. RPMI 1640 medium, fetal bovine serum (FBS), and GlutaMAX were obtained from Gibco. Primary antibody against Iba-1 was purchased from Wako Pure Chemical Industries. Antibody against NF-κB p65 was obtained from Santa Cruz, and myelin basic protein (MBP) was purchased from Proteintech. Alexa Fluor 546-conjugated goat anti-rabbit IgG (H + L) and Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) antibodies were purchased from Invitrogen. A haematoxylin-eosin (HE) staining kit and cell counting kit (CCK)-8 were purchased from Beyotime Biotechnology (Shanghai, China). Human XL Cytokine Array Kit was obtained from R&D.

After transfection for 22–24 h, HEK293 cells were washed and fixed with 2% paraformaldehyde (PFA) in PBS for 12 min, followed by 5 min × 3 washes with PBS. Then cells were blocked with 2% BSA in PBS for 1 h and incubated with mouse monoclonal anti-HA antibody (Millipore, 1:300) or mouse monoclonal anti-Myc antibody (Abcam, 1:300) for 3 h. Cells were washed with PBS 5 times and 1% BSA once. After incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) (Invitrogen, 1:300) for 1–1.5 h, the cells were washed with PBS for 6 times. All the procedures were performed at room temperature (22–25°C).
Fluorescence images were visualized by using a spinning-disk confocal imaging system (CSU-X1 Nipkow Yokogawa, Japan) equipped with an Olympus IX-71 inverted microscope (Olympus Corp., Japan). 16-bit digital images were obtained with oil-immersion objective (100 ×, NA 1.30) and an EM CCD camera (DU897K, ANDOR iXon, United Kingdom).