Biomod/2012/HKBU/BU Magician:Labook:Notebook/Labbook/2012/09/27

From OpenWetWare
Jump to: navigation, search

<html> <style> .container{background-color: #FFFFFF; margin-top:0px}

  1. column-content {width: 0px; float: left; margin: 0 0 0 0;padding: 0;}

.firstHeading {display:none; width:0px;}

  1. column-one {display:none; width:0px;background-color: #FFFFFF;}
  1. globalWrapper{width: 920px; background-color: #FFFFFF; margin-left: auto;margin-right: auto;

-moz-box-shadow: 0px 0px 15px #ccc; -webkit-box-shadow: 0px 0px 15px #ccc; box-shadow: 0px 0px 15px #ccc;}

  1. content{ margin: 0 0 0 0; align: center; padding: 12px 12px 12px 12px; width: 800px;background-color: #FFFFFF; border: 0;}
  2. bodyContent{ width: 800px; align: center; background-color: #FFFFFF;}
  3. column-content{width: 800px;background-color: #FFFFFF;}

</style> <a href="http://openwetware.org/index.php?title=Biomod/2012/HKBU/BU_Magician:Labook:Notebook/Labbook/2012/09/27&action=edit">edit</a> </html> <html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en"> <head> <meta http-equiv="Content-Type" content="text/html; charset=UTF-8" /> <title>jOuery</title> <link rel="stylesheet" href="style.css" /> <style> .menu { height: 45px; display: block; } .menu ul { list-style: none; padding: 0; margin: 0; } .menu ul li { float: left; overflow: hidden; position: relative; text-align: center; line-height: 45px; } .menu ul li a { position: relative; display: block; width: 128px; height: 45px; font-family: Arial; font-size: 11px; font-weight: bold; letter-spacing: 1px; text-transform: uppercase; text-decoration: none; cursor: pointer; } .menu ul li a span { position: absolute; left: 0; width: 128px; } .menu ul li a span.out { top: 0px; } .menu ul li a span.over, .menu ul li a span.bg { top: -45px; }


  1. menu2 { background:#45A8DF; }
  2. menu2 ul li a { color:#FFF; }
  3. menu2 ul li a span.over { background: #A6DD00; color:#333; }
  4. menu2 ul li.nav1 a span.over { background: #fea274; }
  5. menu2 ul li.nav2 a span.over { background: #b0bbba; }
  6. menu2 ul li.nav3 a span.over { background: #a3f091; }
  7. menu2 ul li.nav4 a span.over { background: #86dbf9; }
  8. menu2 ul li.nav5 a span.over { background: #e0caf0; }
  9. menu2 ul li.nav6 a span.over { background: #9dace9; }
  10. menu2 ul li.nav7 a span.over { background: #FFE66F; }

</style> <script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.3/jquery.min.js"></script> <script language="javascript"> $(document).ready(function() {


$("#menu2 li a").wrapInner( '<span class="out"></span>' );

$("#menu2 li a").each(function() { $('<span class="over">' + $(this).text() + '</span>' ).appendTo( this ); });

$("#menu2 li a").hover(function() { $(".out",this).stop().animate({'top':'45px'},200); $(".over",this).stop().animate({'top':'0px'},200);

}, function() { $(".out",this).stop().animate({'top':'0px'},200); $(".over",this).stop().animate({'top':'-45px'},200); });

});

</script> </head> <body> <div id="HEADER">

 <div id="menu2" class="menu" style="width: 897px">
   <ul>
     <li class="nav1"><a href="http://openwetware.org/wiki/Biomod/2012/HKBU/BU_Magician">Home</a></li>
     <li class="nav2"><a href="http://openwetware.org/wiki/Biomod/2012/HKBU/BU_Magician:Projects">Projects</a></li>
     <li class="nav3"><a href="http://openwetware.org/wiki/Biomod/2012/HKBU/BU_Magician:Results">ResultS</a></li>
      <li class="nav7"><a href="http://openwetware.org/wiki/Biomod/2012/HKBU/BU_Magician:Methods">Methods</a></li>
     <li class="nav4"><a href="http://openwetware.org/wiki/Biomod/2012/HKBU/BU_Magician:Team">Team</a></li>
     <li class="nav5"><a href="http://openwetware.org/wiki/Biomod/2012/HKBU/BU_Magician:Labook:Notebook/Labbook">Labook</a></li>
     <li class="nav6"><a href="http://openwetware.org/wiki/Biomod/2012/HKBU/BU_Magician:Extra">Extra</a></li>
   </ul>
 </div>

</div> </body> </html>

2012/09/27

For Plan A: Cell Transferring

(1)Precoat second wells
(2)For treatment with magnetic fields, remove one magnet, and apply only one magnet for 5 s until most of the free nanoparticles are attracted, cling the well to the direction opposite to magnet and transfer medium to a second well. Then add approximately 100ul trypsin to detach the cell on the bottom and transfer to corresponding wells.
(3) Allow growth for 24hrs

For Plan B: Antibody Adding

(1)Primary Antibody Adding
1. Discard all medium in the wells.
2. Washed the cells gently with PBS. For wells with nanoparticles, repeat this step 2 times. For wells without nanoparticles, do this step just once. Discard all PBS.
3. Fix the cells using 3% paraformaldehyde for 30 minutes at room temperature.
4. Washed the cells gently with PBS 3 times.
5. Add primary antibody containing solution(350μl/well).
6. Incubate the cells for 2 hours at room temperature.

Primary antibody containing solution(concentration: 1:1000):
a). NR2B antibody
b). PBS with 0.1% Triton X
c). 2% normal goat serum (NGS)

1ml primary antibody containing solution
= 1μl NR2B antibody + 20ul NGS + 979ul 0.1% TritonX PBS

350μl primary antibody containing solution/well X 14 wells = 4900μl primary antibody containing solution

Primary antibody containing solution preparation:
5ml primary antibody containing solution
= 5μl NR2B antibody + 100ul NGS + 4895ul 0.1% TritonX PBS

(2)Secondary Antibody Adding
7. Discard all primary antibody containing solution.
8. Wash with PBS three times.
9. Add secondary antibody containing solution(350μl/well).
10. Incubate the cells for 2 hours at room temperature.

Secondary antibody:
Fluorescein(Alexa 488)-conjugated goat anti-rabbit secondary antibody

Secondary antibody containing solution(concentration: 1:500):
a). Fluorescein-conjugated goat anti-rabbit secondary antibody
b). PBS

1ml secondary antibody concentration
= 2μl of goat anti rabbit Alexa 488 + 998μl PBS (bench use PBS)

350μl secondary antibody containing solution/well X 14 wells = 4900μl secondary antibody containing solution

Secondary antibody containing solution preparation:
5ml secondary antibody containing solution
=10μl of goat anti rabbit Alexa 488 + 4990μl PBS (bench use PBS)

(3)Slide Preparation:
11. Discard all secondary antibody containing solution and wash with PBS three times.
12. Add one drop mounting medium (DAKO) onto a clean slide.
13. Coverslips with immunostained cells were mounted on the clean slide (without bubbles).