Alexa fluor 568 conjugated goat anti mouse igg

Free merozoites isolated as described above were fixed at RT for 30 min with 4% paraformaldehyde - 0.0075% glutaraldehyde in PBS^{46 (link)}. The merozoites were processed with or without permeabilization with 0.1% Triton X-100 in PBS at RT for 10 min, and blocked with 3% bovine serum albumin (BSA) in PBS at 37 °C for 30 min.
For IFA, the free merozoites were stained with primary antibodies diluted at the following concentrations in blocking solution at 37 °C for 1 h: rabbit anti-PfMSA180-Tr1 antibody, 1:500; rabbit anti- PfMSA180-Tr4, 1:500; mouse anti-MTIP antibody, 1:100; mouse anti-AMA1 antibody, 1:100; and mouse anti-MSP1-19 antibody, 1:100. Secondary antibodies, Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 568-conjugated goat anti-mouse IgG (Invitrogen), were used at a 1:1000 dilution in blocking solution at 37 °C for 30 min. DAPI (4′,6-diamidino-2-phenylindole) at 2 μg/ml was added to the secondary-antibody solution to stain the nuclei. The samples were then applied to polyethyleneimine-coated coverslips. Slides were mounted in ProLong Gold Antifade reagent (Invitrogen) and viewed under a 63× oil immersion lens. High-resolution image capture and processing were performed with confocal scanning laser microscopes (LSM710 and LSM700; Carl Zeiss MicroImaging, Thornwood, NY). Images were processed by Image J (NIH).

IFA was conducted as previously described35 (link). The samples were stained with primary antibodies diluted at the following concentrations in blocking solution at 37 °C for 1 h: human anti-LSA3-C antibody, 0.173 μg/ml; rabbit anti-LSA3-C antibody, 1:500; mouse anti-AMA1 antibody, 1:100; mouse anti-RAP1 antibody, 1:1,000; mouse anti-RON2 antibody, 1:100; mouse anti-RESA monoclonal antibody, 1:100. Secondary antibodies, Alexa Fluor 488-conjugated goat anti-human IgG (Invitrogen, Code # A11013), Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, Code # A11034) and Alexa Fluor 568-conjugated goat anti-mouse IgG (Invitrogen, Code #A11031), were used at a 1:500 dilution in blocking solution at 37 °C for 30 min. DAPI (4’, 6-diamidino-2-phenylindole) (Invitrogen, Code #D1306) at 2 μg/ml was also added to the secondary-antibody solution to stain the nuclei. Slides were mounted in ProLong Gold Antifade reagent (Invitrogen, Code #P36934) and viewed under a 63 × oil immersion lens. High-resolution image capture and processing were performed with a confocal scanning laser microscope (LSM710; Carl Zeiss MicroImaging, Thornwood, NY). Images were processed in Adobe Photoshop (Adobe Systems Inc., San Jose, CA).

hGMSCs in eight-well chamber slides (Merck Millipore) were used for immunocytochemistry. After fixation with 4% paraformaldehyde, cells were permeabilized with methanol, blocked with 5% BSA in PBS for 20 minutes, and then exposed to the primary antibody of nestin, microtubule-associated protein 2 (MAP-2), glial fibrillary acidic protein (GFAP) (Merck Millipore), cbfα-1 (Abcam, Cambridge, UK), or OC (Epitomics Inc., Burlingame, CA, USA) overnight at 4°C. The cells were washed in PBS, exposed to the secondary antibody of FITC-conjugated goat anti-rabbit immunoglobulin G (IgG) (Abcam) or Alexa Fluor 568-conjugated goat anti-mouse IgG (InvitroGen) for 30 minutes and counterstained with 4',6-diamidino-2-phenylindole (DAPI). The nuclear translocation of cbfα-1 and protein expressions of nestin, MAP-2, GFAP and OC in hGMSCs were observed by confocal laser scanning microscopy (LSM780, Carl Zeiss MicroImaging, Inc., New York, NY, USA).

Formalin-fixed paraffin-embedded pancreas tissues were serially sectioned. Consecutive sections were stained for H-E, insulin and glucagon, HA, CD68, CD3, and CD8 in different combinations. Four to six sets of serial sections were prepared per pancreas. All islets present in a stained section, on average 71 islets (range 52–99 islets), were counted per rat. The following primary antibodies were used: insulin (Dako, Glostrup, Denmark), glucagon (Sigma-Aldrich, St Louis, MI, USA), and CD68 (clone ED1, Bio-Rad, Hercules, CA, USA), at dilutions 1:500, 1:2,000, and 1:100, respectively. Antibodies to CD3 (clone SP7, Abcam, Cambridge, UK) and CD8 (Abcam) were used at dilution 1:150. Staining for HA was performed as described (16 (link), 18 (link)). Sections were incubated overnight with the primary antibodies and then for 1 h with the following secondary antibodies at dilution 1:400: Alexa Fluor^® 488 conjugated goat anti-rabbit IgG (Invitrogen), Alexa Fluor^® 568 conjugated goat anti-mouse IgG (Invitrogen), and Cy™2 donkey anti-guinea pig IgG (Jackson Immunoresearch). The stained sections were mounted with VECTASHIELD Vibrance with DAPI (a nuclear counterstain) Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA).

Peritoneal macrophages were plated in a chamber slide and incubated with 500 ng/mL of LPS for 1 h at 37°C. The cells were fixed in 4% paraformaldehyde for 30 min at RT and washed three times with PBS. The cells were treated with 0.1% Triton X-100 for 15 min at room temperature. After washing, the cells were incubated with a blocking serum for 1 h and incubated overnight with a 1 : 100 dilution of primary NF-κB p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The cells were then washed and incubated with a 1 : 2000 dilution of Alexafluor 568-conjugated goat anti-mouse IgG (Invitrogen, USA) for 2 h in a dark room. For nuclear staining, the cells were incubated with a 1 : 1000 dilution of DAPI for 5 min. The slide was finally washed and mounted for microscopic examination. Staining with NF-κB p65 antibody exhibited red fluorescence, which was detected by fluorescence microscopy.

Free merozoites were isolated as reported (17 (link)) and fixed at room temperature (RT) for 30 min with 4% paraformaldehyde/0.0075% glutaraldehyde in PBS. The merozoites were processed with or without permeabilization with 0.1% Triton X-100, and blocked with 3% bovine serum albumin (BSA) in PBS at 37°C for 30 min. The slides were stained with rabbit anti-PfMSP10 antibody, 1:1,000; rabbit anti-PfGAMA, 1:1,000; or mouse anti-PfMTIP antibody, 1:100. Secondary antibodies, Alexa Fluor 488-conjugated goat anti-rabbit IgG, and Alexa Fluor 568-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA), were used at a 1:1,000 dilution at 37°C for 30 min. For nuclei staining, DAPI (4′,6-diamidino-2-phenylindole) at 2 μg/ml was also added. The merozoites were then immobilized on polyethyleneimine-coated coverslips and mounted in ProLong Gold Antifade reagent (Invitrogen). High-resolution image capture and processing were performed with confocal scanning laser microscopes (LSM710 and LSM700; Carl Zeiss MicroImaging, Thornwood, NY) using 63 × oil immersion lens. Images were processed by Image J (NIH).