<?xml version="1.0" encoding="UTF-8"?><ProtocolTitle xmlns="http://morstonmud.com/omero/schemas" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" elementName="&#10;      BubRI staining&#10;    &#10;  " protocolFileName="BubRI-staining.pro.xml" substepsCollapsed="false" version="3.0-3.0" xsi:schemaLocation="http://morstonmud.com/omero/schemas http://morstonmud.com/omero/schemas/PE.xsd"><DateTimeField UTCMillisecs="1206316800000" alarmSeconds="-3600" description="&#10;  " elementName="BubRI staining" seconds="54000"/><TextBox elementName="&#10;      Background, &#10;    &#10;    &lt;br&gt;&#10;      reason for experiment&#10;    &#10;  " value="Want to look at the effect on BubRI of knocking down CSFV etc. &#10;Need good staining of BubRI (mouse antibody)."/><TextBox displayChildrenHorizontally="true" elementName="&#10;      Summary of Experiment&#10;    &#10;  " substepsCollapsed="false" value="Carry out siRNAi and then stain for BubRI and other markers.&#10;Need to use methanol/acetone fixation to maintain BubRI epitopes."/><FixedStep elementName="&#10;      Methods&#10;    &#10;  " substepsCollapsed="false"><DropDownMenu default="24-well plate" description="&#10;      Cells are grown on coverslips in dish, except for MatTech.&#10;    &#10;  " dropdownOptions="10cm dish, 6cm dish, 35mm MatTech, 6-well plate, 24-well plate, 96-well plate" elementName="Dish / plate format" value="6-well plate"/><ProtocolTitle description="Manufacturer's protocol for transfecting HeLa cells" elementName="Oligofectamine-Invitrogen" fieldLockedUTC="1208153998355" fieldLockedUserName="will" lockLevel="templateLocked" protocolFileName="Oligofectamine-Invitrogen.pro" protocolHasExpChildren="false" substepsCollapsed="false"><FixedStep description="Oligofectamine has been used to transfect HeLa successfully (Elbashir etal 2001).&#10;The suggested (default) transfection conditions are provided as a starting point.&#10;&#10;" elementName="Introduction" substepsCollapsed="false"/><FixedStep description="Transfection Efficiency&#10;Transcription rate of gene of interest&#10;Protein Stability&#10;Efficicacy of the siRNA sequence used&#10;Cell line growth characteristics&#10;" elementName="Factors Affecting Knockdown Efficiency" substepsCollapsed="false"/><FixedStep elementName="General Guidelines" substepsCollapsed="true"><FixedStep elementName="Confluence 30-50%" substepsCollapsed="false"/><FixedStep elementName="Don't use antibiotics" substepsCollapsed="false"/><FixedStep description="Reduced serum medium (Cat no. 31985-062) to dilute Oligofectamine prior to complexing with siRNA." elementName="Use Opti-MEM I" substepsCollapsed="false"/></FixedStep><FixedStep elementName="Materials Needed" substepsCollapsed="true"><FixedStep description="or other mammalian cells. Must be healthy &gt;90% viable before transfection" elementName="HeLa cells" substepsCollapsed="false"/><FixedStep elementName="siRNAi (20 pmol / ul)" substepsCollapsed="false"/><FixedStep elementName="Oligofectamine" substepsCollapsed="false"/><FixedStep elementName="Opti-MEM I" substepsCollapsed="false"/><FixedStep elementName="24-well tissue culture plates" substepsCollapsed="false"/></FixedStep><FixedStep elementName="Conditions" substepsCollapsed="false"><NumberField default="50" description="30,000 cells per well is 50% confluent in 24-well plate" elementName="Cell density" substepsCollapsed="false" units="%" value="75"/><NumberField default="3" elementName="Amount of Oligofectamine" substepsCollapsed="false" units="ul" value="5"/><NumberField default="60" description="Stock should be 20 pmol/ul" elementName="Amount of siRNA" substepsCollapsed="false" units="pmol" value="20"/><DateTimeField UTCMillisecs="172800000" alarmSeconds="-10800" elementName="Duration of transfection"/></FixedStep><FixedStep elementName="Proceedure" substepsCollapsed="true"><FixedStep description="One day before transfection, seed HeLa so they will be aprox 50% confluent at time of transfection" elementName="Plate HeLa" substepsCollapsed="false"/><FixedStep elementName="For each sample..." substepsCollapsed="true"><FixedStep description="mix gently" elementName="Dilute siRNA in 50 ul Opti-MEM" substepsCollapsed="false"/><FixedStep description="Incubate for 5 mins at room temp" elementName="Dilute 3ul Oligofectamine in 12 ul OptiMEM" substepsCollapsed="false"/><FixedStep description="Combine the two mixtures to give total volume of 68 ul. Incubate 20 mins at room temp" elementName="Mix siRNAi and Oligofectamine" substepsCollapsed="false"/></FixedStep><FixedStep description="Mix gently by rocking plate back and forwards" elementName="Add siRNAi:Oligofectamine to each well" substepsCollapsed="false"/><FixedStep description="in CO2 incubator for 24-72 hrs. Not usually necessary to remove complexes or change medium. However, can replace medium after 4-6 hours without loss of transfection activity." elementName="Incubate" substepsCollapsed="false"/></FixedStep></ProtocolTitle><DateTimeField UTCMillisecs="0" alarmSeconds="-3600" elementName="Fix cells" seconds="46800"/><FixedStep elementName="&#10;      Methanol/Acetone fixation&#10;    &#10;  " substepsCollapsed="true"><FixedStep elementName="&#10;      Use meth/acetone 1:1&#10;    &#10;  "/><FixedStep elementName="&#10;      Filled wells of 6-well plate with MeOH/acetone in freezer&#10;    &#10;  "/><FixedStep elementName="&#10;      Picked coverslips out of 24-well plate, dip into PBS &#10;    &#10;    &lt;br&gt;&#10;      and into MeOH/acet in TC hood 2 mins&#10;    &#10;  "/><FixedStep elementName="&#10;      Took off MeOH/acet and washed in PBS x2&#10;    &#10;  "/><FixedStep elementName="&#10;      Block and stain&#10;    &#10;  "/></FixedStep><FixedStep description="This protocol describes immunostaining of fixed and permeabilised cells" elementName="Immuno-Staining" substepsCollapsed="true"><FixedStep description="Place coverslips in moist chamber, on parafilm, cell side up. Keep moist with wet tissues in chamber." elementName="Prepare chamber" substepsCollapsed="false"/><TimeField default="7200" description="Block with 1% NDS/Abdil (spin before use) for 30-60 mins." elementName="Block with Abdil" seconds="7200"/><TimeField default="3900" description="Antibodies are diluted in Abdil. Primary antibodies from different hosts can be mixed and incubated together. 30-60 minutes on cells." elementName="Primary Antibody Incubation" seconds="3900"/><FixedStep description="Wash 3 times with TBS-0.1% Triton X-100.&#10;Wash is at least 3.5ml TBSTx, applied to one side of the coverslip and aspirated off the other side, so as to flow gently across the coverslip. Don't let them dry out. &#10;Repeat 3 times. &#10;Let sit for 3-5 mins." elementName="Wash with TBS-Tx100" substepsCollapsed="false"/><FixedStep description="Secondary antibody glycerol stocks are usually diluted 1:150 in Abdil. Incubate on coverslips for 30 mins.&#10;Wash as before with TBSTx." elementName="Secondary Ab &amp; Wash" substepsCollapsed="false"/><FixedStep description="Wash 3x with TBS. Coverlips will dry quickly. Don't let coverslips dry out!&#10;If using DAPI, incubate with 1ug/ml DAPI for 5 mins in TBS.&#10;Wash with TBS twice. &#10;Dab off xs TBS and mount onto a drop of mounting medium. Seal. " elementName="Wash TBS" substepsCollapsed="false"/></FixedStep><TextField description="&#10;      The target proteins that are to be highlighted by immunofluorescence.&#10;    &#10;  " elementName="&#10;      Staining for &#10;    &#10;  " value="INCENP, microtubules, ACA "/></FixedStep><FixedStep elementName="&#10;      Results&#10;    &#10;  " substepsCollapsed="true"><TableField elementName="Mitotic cells expressing BubRI" rowNumber0="scrambled, 43, 41" rowNumber1="INCENP, 35, 5" rowNumber2="CSFV, 50, 39" rowNumber3="VRAQ, 31, 25" rowNumber4=", , " tableColumnNames="siRNAi, total cells, BubRI stain" tableRowCount="5"/></FixedStep><TextBox elementName="&#10;      Conclusions&#10;    &#10;  " value="BubRI stain worked well (some background).&#10;Chromatin structure not so good with MeOH/acetone. Varied between coverslips. &#10;  Mitotics easy to find on some coverslips but not on others.&#10;CSFV stain didn't work very well. But still can see difference between Scrambled siRNAi and CSFV.&#10;Aurora-B stain OK.&#10;Borealin stain of the same cells not good in metaphse but OK in anaphase/telophase."/></ProtocolTitle>