Staining:Article Title: Myocardial Delivery of Lipidoid Nanoparticle Carrying modRNA Induces Rapid and Transient Expression
Article Snippet: Fluorescent images were obtained using a digital epifluorescent microscope (EVOSfl, Life Technologies). .. Pig heart samples were treated with the following primary antibodies: mouse monoclonal anti-GFP (1:100, 632375, Clontech) rabbit anti-α-actinin (1:50, SC15335, Santa Cruz) chicken anti-vimentin (1:100, ab24525, abcam), mouse anti-α-smooth muscle actin (1:100, A5228, Sigma) mouse anti-pig macrophages (1:50, MCA2317GA, AbD Serotec) and rabbit polyclonal anti-GFP (1:100, 632592, Clontech) (this polyclonal antibody was used for the dual staining with the mouse anti-α-smooth muscle actin and the mouse anti-pig macrophages antibodies, in all other instances, the mouse monoclonal anti-GFP listed above was used); and secondary antibodies: donkey anti-mouse AF 555 (1:300, {"type":"entrez-protein","attrs":{"text":"A31570","term_id":"85652","term_text":"pir||A31570"}} A31570 , Life Technologies), goat anti-rabbit AF 488 (1:300, {"type":"entrez-nucleotide","attrs":{"text":"A11034","term_id":"489250","term_text":"A11034"}} A11034 , Life Technologies), goat anti-chicken AF 488 (1:200, A-11039, Life Technologies), donkey anti-mouse AF 488 (1:200, {"type":"entrez-nucleotide","attrs":{"text":"A21202","term_id":"641355","term_text":"A21202"}} A21202 , Life Technologies), and donkey anti-rabbit AF 546 (1:200, {"type":"entrez-nucleotide","attrs":{"text":"A10040","term_id":"489103","term_text":"A10040"}} A10040 , Life Technologies); for isotype control, we used mouse mAB IgG1 (1:125, 5415, Cell signaling); nuclei were stained with DAPI. .. Fluorescent images were obtained using a laser scanning confocal microscope (Leica TCS SP5 DMI, Leica Microsystems, Buffalo Grove, IL), a digital epifluorescent microscope (EVOSfl, Life Technologies), and a Zeiss Axioplan2IE with ApoTome structured illumination technology.
Article Title: Heat stress induced testicular impairment is related to orchitis and complement activation in Rongchang boars.
Article Snippet: .. Cells were later resuspended in 500 μL of Cytoperm (BD, Franklin Lakes, New Jersey, USA) to permeate the cell membrane for 5 min. After centrifugation at 350 × g for 5 min at 4 °C and the disposal of Cytoperm, 100 μL of Cytoperm containing mouse anti-pig macrophages: Alexa Fluor 647 (Bio-Rad, Hercules, California, USA) was added to resuspend the cells at 4 °C for 40 min. Lastly, each cell suspension was centrifuged at 350 × g and 4 °C to remove the supernatant and washed once with 100 μL Stain Buffer. .. Then, 350 μL of Stain Buffer was used to resuspend the cells for analysis using a BD Verse flow cytometer (BD, Franklin Lakes, New Jersey, USA).
Article Title: Heat stress induced testicular impairment is related to orchitis and complement activation in Rongchang boars
Article Snippet: .. Cells were later resuspended in 500 μL of Cytoperm (BD, Franklin Lakes, New Jersey, USA) to permeate the cell membrane for 5 min. After centrifugation at 350 × g for 5 min at 4 °C and the disposal of Cytoperm, 100 μL of Cytoperm containing mouse anti-pig macrophages: Alexa Fluor 647 (Bio-Rad, Hercules, California, USA) was added to resuspend the cells at 4 °C for 40 min. Lastly, each cell suspension was centrifuged at 350 × g and 4 °C to remove the supernatant and washed once with 100 μL Stain Buffer. .. Then, 350 μL of Stain Buffer was used to resuspend the cells for analysis using a BD Verse flow cytometer (BD, Franklin Lakes, New Jersey, USA).
Control:Article Title: Myocardial Delivery of Lipidoid Nanoparticle Carrying modRNA Induces Rapid and Transient Expression
Article Snippet: Fluorescent images were obtained using a digital epifluorescent microscope (EVOSfl, Life Technologies). .. Pig heart samples were treated with the following primary antibodies: mouse monoclonal anti-GFP (1:100, 632375, Clontech) rabbit anti-α-actinin (1:50, SC15335, Santa Cruz) chicken anti-vimentin (1:100, ab24525, abcam), mouse anti-α-smooth muscle actin (1:100, A5228, Sigma) mouse anti-pig macrophages (1:50, MCA2317GA, AbD Serotec) and rabbit polyclonal anti-GFP (1:100, 632592, Clontech) (this polyclonal antibody was used for the dual staining with the mouse anti-α-smooth muscle actin and the mouse anti-pig macrophages antibodies, in all other instances, the mouse monoclonal anti-GFP listed above was used); and secondary antibodies: donkey anti-mouse AF 555 (1:300, {"type":"entrez-protein","attrs":{"text":"A31570","term_id":"85652","term_text":"pir||A31570"}} A31570 , Life Technologies), goat anti-rabbit AF 488 (1:300, {"type":"entrez-nucleotide","attrs":{"text":"A11034","term_id":"489250","term_text":"A11034"}} A11034 , Life Technologies), goat anti-chicken AF 488 (1:200, A-11039, Life Technologies), donkey anti-mouse AF 488 (1:200, {"type":"entrez-nucleotide","attrs":{"text":"A21202","term_id":"641355","term_text":"A21202"}} A21202 , Life Technologies), and donkey anti-rabbit AF 546 (1:200, {"type":"entrez-nucleotide","attrs":{"text":"A10040","term_id":"489103","term_text":"A10040"}} A10040 , Life Technologies); for isotype control, we used mouse mAB IgG1 (1:125, 5415, Cell signaling); nuclei were stained with DAPI. .. Fluorescent images were obtained using a laser scanning confocal microscope (Leica TCS SP5 DMI, Leica Microsystems, Buffalo Grove, IL), a digital epifluorescent microscope (EVOSfl, Life Technologies), and a Zeiss Axioplan2IE with ApoTome structured illumination technology.
Membrane:Article Title: Heat stress induced testicular impairment is related to orchitis and complement activation in Rongchang boars.
Article Snippet: .. Cells were later resuspended in 500 μL of Cytoperm (BD, Franklin Lakes, New Jersey, USA) to permeate the cell membrane for 5 min. After centrifugation at 350 × g for 5 min at 4 °C and the disposal of Cytoperm, 100 μL of Cytoperm containing mouse anti-pig macrophages: Alexa Fluor 647 (Bio-Rad, Hercules, California, USA) was added to resuspend the cells at 4 °C for 40 min. Lastly, each cell suspension was centrifuged at 350 × g and 4 °C to remove the supernatant and washed once with 100 μL Stain Buffer. .. Then, 350 μL of Stain Buffer was used to resuspend the cells for analysis using a BD Verse flow cytometer (BD, Franklin Lakes, New Jersey, USA).
Article Title: Heat stress induced testicular impairment is related to orchitis and complement activation in Rongchang boars
Article Snippet: .. Cells were later resuspended in 500 μL of Cytoperm (BD, Franklin Lakes, New Jersey, USA) to permeate the cell membrane for 5 min. After centrifugation at 350 × g for 5 min at 4 °C and the disposal of Cytoperm, 100 μL of Cytoperm containing mouse anti-pig macrophages: Alexa Fluor 647 (Bio-Rad, Hercules, California, USA) was added to resuspend the cells at 4 °C for 40 min. Lastly, each cell suspension was centrifuged at 350 × g and 4 °C to remove the supernatant and washed once with 100 μL Stain Buffer. .. Then, 350 μL of Stain Buffer was used to resuspend the cells for analysis using a BD Verse flow cytometer (BD, Franklin Lakes, New Jersey, USA).
Centrifugation:Article Title: Heat stress induced testicular impairment is related to orchitis and complement activation in Rongchang boars.
Article Snippet: .. Cells were later resuspended in 500 μL of Cytoperm (BD, Franklin Lakes, New Jersey, USA) to permeate the cell membrane for 5 min. After centrifugation at 350 × g for 5 min at 4 °C and the disposal of Cytoperm, 100 μL of Cytoperm containing mouse anti-pig macrophages: Alexa Fluor 647 (Bio-Rad, Hercules, California, USA) was added to resuspend the cells at 4 °C for 40 min. Lastly, each cell suspension was centrifuged at 350 × g and 4 °C to remove the supernatant and washed once with 100 μL Stain Buffer. .. Then, 350 μL of Stain Buffer was used to resuspend the cells for analysis using a BD Verse flow cytometer (BD, Franklin Lakes, New Jersey, USA).
Article Title: Heat stress induced testicular impairment is related to orchitis and complement activation in Rongchang boars
Article Snippet: .. Cells were later resuspended in 500 μL of Cytoperm (BD, Franklin Lakes, New Jersey, USA) to permeate the cell membrane for 5 min. After centrifugation at 350 × g for 5 min at 4 °C and the disposal of Cytoperm, 100 μL of Cytoperm containing mouse anti-pig macrophages: Alexa Fluor 647 (Bio-Rad, Hercules, California, USA) was added to resuspend the cells at 4 °C for 40 min. Lastly, each cell suspension was centrifuged at 350 × g and 4 °C to remove the supernatant and washed once with 100 μL Stain Buffer. .. Then, 350 μL of Stain Buffer was used to resuspend the cells for analysis using a BD Verse flow cytometer (BD, Franklin Lakes, New Jersey, USA).
Suspension:Article Title: Heat stress induced testicular impairment is related to orchitis and complement activation in Rongchang boars.
Article Snippet: .. Cells were later resuspended in 500 μL of Cytoperm (BD, Franklin Lakes, New Jersey, USA) to permeate the cell membrane for 5 min. After centrifugation at 350 × g for 5 min at 4 °C and the disposal of Cytoperm, 100 μL of Cytoperm containing mouse anti-pig macrophages: Alexa Fluor 647 (Bio-Rad, Hercules, California, USA) was added to resuspend the cells at 4 °C for 40 min. Lastly, each cell suspension was centrifuged at 350 × g and 4 °C to remove the supernatant and washed once with 100 μL Stain Buffer. .. Then, 350 μL of Stain Buffer was used to resuspend the cells for analysis using a BD Verse flow cytometer (BD, Franklin Lakes, New Jersey, USA).
Article Title: Heat stress induced testicular impairment is related to orchitis and complement activation in Rongchang boars
Article Snippet: .. Cells were later resuspended in 500 μL of Cytoperm (BD, Franklin Lakes, New Jersey, USA) to permeate the cell membrane for 5 min. After centrifugation at 350 × g for 5 min at 4 °C and the disposal of Cytoperm, 100 μL of Cytoperm containing mouse anti-pig macrophages: Alexa Fluor 647 (Bio-Rad, Hercules, California, USA) was added to resuspend the cells at 4 °C for 40 min. Lastly, each cell suspension was centrifuged at 350 × g and 4 °C to remove the supernatant and washed once with 100 μL Stain Buffer. .. Then, 350 μL of Stain Buffer was used to resuspend the cells for analysis using a BD Verse flow cytometer (BD, Franklin Lakes, New Jersey, USA).
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