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After incubation with the antibody/complement mixture, the cells were washed twice with MEM and cultivated under neurosphere forming conditions at a clonal density of 1250 cells/ml. When immunocytolysis was performed with cells from mixed neural cultures, the latter were grown to confluence for 6 ? days and subsequently were incubated with 0.05  Trypsin/EDTA (Invitrogen) until the cells detached from the cell culture dish to obtain a single cell suspension. The cells were then diluted in MEM with 0.2  BSA, and immunocytolysis was performed as described for the acutely dissociated cells from E14.5 cortex. Following immunocytolysis, the cells were plated on poly-D-lysin (Sigma) coated cell culture dishes (Nunc, Wiesbaden, Germany) for 24 h in DMEM supplemented with 10  (v/v) horse serum before analysis. Transfection of COS7 Cells--COS7 cells were routinely cultured on uncoated 10-cm dishes at 37  , 6  CO2 in DMEM supplemented with 10  (v/v) FCS, 100 units/ml penicillin, and 100 g/ml streptomycin. Lipofection was performed according to the manufacturer's protocol using the Lipofectamine 2000 reagent (Invitrogen). The cells were transfected with a fucosyltransferase 9 (Fut9) expression plasmid, containing full-length mouse Fut9 cloned into pcDNA3.1 or the empty pcDNA3.1 vector (Invitrogen) as a negative control. To identify transfected cells by GFP fluorescence, the cells were cotransfected with pEGFP-N1 (Invitrogen; ratio pcDNA3.1/pEGFP-N1 5:1). Immunocyto- and Immunohistochemistry--Immunocytochemical staining was performed as outlined previously (27). For staining of acutely dissociated cells, cells from embryonic tissue were resuspended in neurosphere medium and plated on Laminin-coated dishes for 2 h before immunocytochemical analysis was performed. The cells from postnatal tissue were grown in DMEM (Invitrogen) 10  FCS on Laminin for 24 h before analysis. For simultaneous staining of mAb 5750 withMAY 6, 2011 ?VOLUME 286 ?NUMBERmAbs 487LeX and MMALeX, 5750 antibody was linked to NHSPEO4-Biotin (AppliChem), and stainings were performed sequentially using streptavidin Cy3 (Dianova) conjugate. For immunohistochemistry, E13.5 or E14.5 embryos were removed, and immersion was fixed for 8 h with 4  (w/v) paraformaldehyde in PBS at 4  . Tissues were cryoprotected overnight with 20  (w/v) sucrose in PBS, embedded in tissuefreezing medium (Jung, Nussloch, Germany), and frozen on dry ice. The sections (14 ?6 m) were cut on a cryostat (Leica, Solms, Germany). Before immunostaining, cryosections were rehydrated in blocking solution (PBS/10  FCS) for 2 h at room temperature in a humidified slide chamber. Primary antibodies were diluted in blocking solution and incubated overnight at 4  . The sections were washed three times for 5 min with PBS containing 0.1  BSA (PBS/A). Secondary antibodies were diluted in PBS/A and applied for 2 h in the presence of Hoechst (1:100,000). After three final washing steps with PBS, the cryosections were mounted with glass coverslips in ImmuMount. Glycanase [https://britishrestaurantawards.org/members/redcandle3/activity/360534/ https://britishrestaurantawards.org/members/redcandle3/activity/360534/] Digestion--L1-CAM, Tenascin-C, and Phosphacan were purified from mouse brains as previously described (23, 24). 300 g of affinity-purified protein were adjusted to a final concentration of 0.4  (w/v) SDS and 2  (v/v) -mercaptoethanol and a volume of 40 l in PBS. The samples were boiled for 5 min at 95  . After cooling on ice, the denatured samples were diluted 1:1 with N-glycosidase F buffer (150 mM Na2HPO3 pH 8, 50 mM EDTA, 3  (v/v) Nonidet P-40) before addin.
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1. Establish Clear Objectives
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When you set up a clear objective, your audience can focus its energy and attention. Once set, those activities you undertake within your web meeting really should secure the achievement of your respective chosen goals.
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When you determine goal, you send a graphic towards the subconscious mind of your respective attendees. It remains there prior to the objective becomes reality. This is how all creative people work - they transform the images they seed of their subconscious minds in a concrete form.
 +
Henceforth, only items in support of your objectives are mentioned inside meeting. Examples of objectives that may be established are: "Discovery session of how our widget can solve the issue of... ", or "Exploration of the various uses of the widget", etc.
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 2. Prepare
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Today's modern web conferencing systems permit the presenter to only invite and accept attendees. But that's only the beginning. Make sure you are informed about the functions that you will have to have a great web meeting.
 +
The basic 8 functions you may need are: desktop sharing, participant list/control, drawing tools, remote keyboard and mouse control, text chat, session recording, VoIP (for sending sound from a PC on the attendees), and polling/surveys. (These are the absolute minimum requirements - if you don't have these traits you will need to find another vendor). Read on to find how you'll use these tools.
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3. Provide Visuals (a.k.a. channeling your "inner Steve Jobs")
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Does this problem? You're interested in a new widget and obtain invited to a web site demo by the Widget Company. The presenter at WidgetCo opens up his stock PowerPoint presentation, goes through it slide by boring slide, then asks you for the P.O. or plastic card number.
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P-L-E-A-S-E. Take advantage of this powerful technology. Make your presentation incredibly compelling. There's nothing wrong with PowerPoint - along with "kill them" by using it. Use a number of [https://www.livestreamingsydney.com.au/ Live streaming Sydney] visually appealing PowerPoint slides to establish and interactively highlight the key points. Then, draw out the widget. Pass the controls for the audience to allow them to "touch", "smell", and "taste" the widget. Start the fog machine. Mix in some audio and video. Go to your a way to imply to them best places to learn more. Are you receiving the idea?
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4. Interact using your audience
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Solicit questions from your attendees. Check in often to gauge the eye level. Unlike an actual physical meeting you simply can't read the mannerisms of your participants. Pay attention to tiny problems, such as the breathing (or snoring) alternatively. Remember your established objective and remain focused. Fail to achieve this at your peril - maybe you have lost the sale before you even understand it.
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5. Follow up
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Offer attendees a meeting summary consisting in the presentation slides, documents, session recording for later review, and chat logs. Afterward - conduct polls, evaluations, and even quizzes. Finally - send thank-you notes, solicit additional questions and establish the following steps.
 +
In summary, your next online web meeting can be a success, providing it's planned well, properly executed, compelling, and focused. Stay in contact together with your participants despite your session ends, as well as your web meeting could have proven to be a hit.

รุ่นแก้ไขเมื่อ 01:25, 10 ธันวาคม 2564

1. Establish Clear Objectives When you set up a clear objective, your audience can focus its energy and attention. Once set, those activities you undertake within your web meeting really should secure the achievement of your respective chosen goals. When you determine goal, you send a graphic towards the subconscious mind of your respective attendees. It remains there prior to the objective becomes reality. This is how all creative people work - they transform the images they seed of their subconscious minds in a concrete form. Henceforth, only items in support of your objectives are mentioned inside meeting. Examples of objectives that may be established are: "Discovery session of how our widget can solve the issue of... ", or "Exploration of the various uses of the widget", etc.  2. Prepare Today's modern web conferencing systems permit the presenter to only invite and accept attendees. But that's only the beginning. Make sure you are informed about the functions that you will have to have a great web meeting. The basic 8 functions you may need are: desktop sharing, participant list/control, drawing tools, remote keyboard and mouse control, text chat, session recording, VoIP (for sending sound from a PC on the attendees), and polling/surveys. (These are the absolute minimum requirements - if you don't have these traits you will need to find another vendor). Read on to find how you'll use these tools. 3. Provide Visuals (a.k.a. channeling your "inner Steve Jobs") Does this problem? You're interested in a new widget and obtain invited to a web site demo by the Widget Company. The presenter at WidgetCo opens up his stock PowerPoint presentation, goes through it slide by boring slide, then asks you for the P.O. or plastic card number. P-L-E-A-S-E. Take advantage of this powerful technology. Make your presentation incredibly compelling. There's nothing wrong with PowerPoint - along with "kill them" by using it. Use a number of Live streaming Sydney visually appealing PowerPoint slides to establish and interactively highlight the key points. Then, draw out the widget. Pass the controls for the audience to allow them to "touch", "smell", and "taste" the widget. Start the fog machine. Mix in some audio and video. Go to your a way to imply to them best places to learn more. Are you receiving the idea? 4. Interact using your audience Solicit questions from your attendees. Check in often to gauge the eye level. Unlike an actual physical meeting you simply can't read the mannerisms of your participants. Pay attention to tiny problems, such as the breathing (or snoring) alternatively. Remember your established objective and remain focused. Fail to achieve this at your peril - maybe you have lost the sale before you even understand it. 5. Follow up Offer attendees a meeting summary consisting in the presentation slides, documents, session recording for later review, and chat logs. Afterward - conduct polls, evaluations, and even quizzes. Finally - send thank-you notes, solicit additional questions and establish the following steps. In summary, your next online web meeting can be a success, providing it's planned well, properly executed, compelling, and focused. Stay in contact together with your participants despite your session ends, as well as your web meeting could have proven to be a hit.

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