Alexafluor 488 f ab 2 fragment of goat anti mouse igg h l

Monolayer cultures of hTERT-HSC were fixed in 4% paraformaldehyde (PFA) for 15 minutes, followed by permeabilization with 0.1% Triton-X-100 for 20 minutes. Blocking was performed with 1% BSA in PBS for 60 minutes and washing with PBS; all steps were performed at RT. Primary antibody against αSMA (Sigma, A5228, dilution 1:200) and secondary antibody Alexa Fluor^® 488 F(ab')2 Fragment of Goat Anti-Mouse IgG (H+L) (Invitrogen, A11017, dilution 1:400) were used for the staining.

Immunofluorescence assays (IFAs) were performed using air-dried sporozoites or ookinetes obtained from mosquito midguts dissected 24 h after feeding on an infectious blood meal. Briefly, a sporozoite suspension (4 × 10⁵ to 6 × 10⁵ sporozoites/ml) or ookinete suspension was air dried at room temperature on poly-l-lysine-coated slides (Tekdon Inc., Myakka City, FL). Parasites were fixed for 30 min using a 2% (wt/vol) paraformaldehyde (Sigma, St. Louis, MO)–1× phosphate-buffered saline (PBS) solution and washed 2 times. Sporozoites were then permeabilized for 30 min using a 0.1% (vol/vol) Triton (Roche, Mannheim, Germany)–1× PBS solution and washed. Antibody or serum samples diluted in 1% (wt/vol) bovine serum albumin (BSA) (Sigma, St. Louis, MO)–1× PBS (1% BSA–PBS) were incubated for 30 min at room temperature. Slides were then washed with 1% BSA–PBS and incubated for 30 min at room temperature with a secondary-antibody solution [Alexa Fluor 488 F(ab′)₂ fragment of goat anti-mouse IgG(H+L); 2 mg/ml; (Invitrogen)]. Fluorescent sporozoites and ookinetes were visualized under an upright fluorescence microscope (Nikon Eclipse 90i).

Antibodies against the following proteins were used in this study: GAPDH (Santa Cruz, Dallas, TX, USA), LAMIN B1 (MBL, Tokyo, Japan), PARIS (Millipore), PARIS (Proteintech, Chicago, IL, USA), PGC-1α (Millipore), PDGFRα (R&D, Minneapolis, MN, USA), PPARγ (Santa Cruz) HRP-conjugated F(ab’)2 fragment of goat anti-rabbit IgG (Jackson ImmunoResearch), HRP-conjugated F(ab’)2 fragment of goat anti-mouse IgG (Jackson ImmunoResearch), HRP-conjugated F(ab’)2 fragment of donkey anti-goat IgG (Jackson ImmunoResearch), Alexa Fluor® 594 F(ab’)2 fragment of goat anti-mouse IgG (Invitrogen, Waltham, MA, USA), Alexa Fluor™ 488 F(ab’)2 fragment of goat anti-mouse IgG (H + L) (Invitrogen), and Alexa Fluor™ 594 F(ab’)2 fragment of goat anti-rabbit IgG (H + L) (Invitrogen).

U937 cells were differentiated with 20 nM PMA or DMSO (control) at 5×10⁵ cells/ml/well for 24–72 h under normal cell culture conditions. At each time point 4 wells were washed once with cold PBS and dislodged with Accutase 200µl/well for 30 minutes. 5×10⁵cells/tube were washed twice with 500µl/tube cold Hepes buffer containing 3% BSA. Cells were blocked with 50µl buffer containing Human TruStain FcX (50µl/ml, Biolegend, San Diego, CA, USA) for 10 minutes at RT. After blocking, 50µl monoclonal anti PCI antibody (4PCI; Technoclone, Vienna, Austria) or mouse monoclonal IgG1 isotype control (R&D Systems, Minneapolis, MN, USA) were added to reach a final concentration of 30µg/ml and incubated for 1 h RT. Cells were washed twice and incubated with 50µl secondary Alexa Fluor 488 F(ab')2 fragment of goat anti-mouse IgG (H+L) (diluted 1∶1000, Invitrogen, Life Technologies, Carlsbad, CA, USA) for 45 minutes at RT. After washing, cells were fixed in 1% PFA and stored in the dark until FACS analysis. PCI detection was quantified by the mean fluorescent intensity multiplied with the percentage of Alexa 488 positive cells. The isotype control was substracted.

50 μL of E. coli cell culture (at ~1x10⁹ cells/mL) that recombinantly expresses Chlamydia MOMP was incubated with 50 μL of mouse sera generated in-house against Chlamydia EBs at a dilution of 1:250 for 1 h at room temperature in a 96 well plate. After incubation, the cells were washed with 1 mL phosphate buffered saline (PBS) and stained with 100 μL of a fluorescence labeled secondary antibody (Alexa Fluor-488 F(ab)’2 fragment of goat anti-mouse IgG (H + L), Life Technologies) at a dilution of 1:100. The stained cells were washed twice and re-suspended in PBS for flow cytometric analysis (Guava Technologies). Data analyses were performed with CytoSoft 5.3 software (Guava Technologies).

Monoclonal mouse to Tn (5F4, IgM), mouse to T (3C9, IgM), mouse to STn (3F1, IgG), mouse to GalNAc-T2 (4C4, IgG) mouse to β4Gal-T1 (2F5, IgG) and polyclonal rabbit to gC-1 (KF922, 1:700) antibodies were produced as previously described [24 (link), 91 (link)]. Rabbit anti-giantin (1:500) and rat anti-GRP94 (1:50) were purchased from Abcam. FITC-conjugated HPA (Helix pomatia agglutinin, 1:2000) was from Invitrogen. FITC-conjugated polyclonal rabbit anti-HSV-1 antibody was purchased from DAKO (1:40). Alexa Fluor 488 F(ab')₂ fragment of Goat anti-Mouse IgG (H+L) (1:500), Alexa Fluor 546 Goat anti-Mouse IgM (μ chain) (1:500) were from Life Technologies. FITC-conjugated polyclonal Goat anti-Mouse antibody (1:100) and TRITC-conjugated Swine anti-Rabbit antibody (1:200) were from DAKO. Alexa Fluor 647 Goat anti-Mouse IgM (μ chain) was purchased from Life Technologies.