<?xml version="1.0" encoding="UTF-8"?><ProtocolTitle xmlns="http://www.openmicroscopy.org/Schemas/Editor/2008-04" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" elementName="Title - click to edit" protocolFileName="OntologiesExample.pro.xml" version="3.0-3.0" xsi:schemaLocation="http://www.openmicroscopy.org/Schemas/Editor/2008-04 http://www.openmicroscopy.org/Schemas/Editor/2008-04/Editor.xsd"><ProtocolTitle elementName="Fix-Stain-4channel" protocolFileName="AuroraB fix-stain.exp" substepsCollapsed="true" version="1.0" xsi:schemaLocation="http://morstonmud.com/omero/schemas http://morstonmud.com/omero/schemas/PE.xsd"><DateField default="Sep 24, 2007" elementName="Fixation Date" substepsCollapsed="false" value="Sep 21, 2007"/><TextBox elementName="Comments" substepsCollapsed="false" value="Looking for localisation of Aurora-B at the inner centromere and midbody of mitotic HeLa cells.&#10;Trying a weak Aurora-B antibody, so tried staining overnight."/><FixedStep description="This protocol describes fixing of adherent cells with formaldehyde" elementName="PFA Fixation" substepsCollapsed="true"><FixedStep description="Make fresh formaldehyde. Mix 3.5ml ddH2O, 1.85g Merck paraformaldehyde and 10ul 10 N KOH in a 50ml tube. Boil water in beaker in microwave, place tube in water." elementName="Prepare PFA" substepsCollapsed="false"/><FixedStep description="Let PFA mixture cool (5 mins) and then filter through 0.2um filter. This is 37% PFA.&#10;Dilute this in 1 x PBS. Usually dilute 10x to 3.7%" elementName="Filter &amp; Dilute PFA" substepsCollapsed="false"/><FixedStep description="Take medium  off cells and wash coverslips by dipping 3 times in PBS" elementName="Wash coverslips in buffer" substepsCollapsed="false"/><FixedStep description="Immediately add coverslips to the freshly diluted PFA. Swirl every 1-2 mins. Incubate for 5 mins." elementName="Add coverslips to PFA" substepsCollapsed="false"/><FixedStep description="Aspirate off PFA. Add another bolus for another 5 mins." elementName="Remove and add more PFA" substepsCollapsed="false"/><FixedStep description="Aspirate off PFA. Wash coverslips 2x with 0.1% Triton X-100 in PBS. Let them sit in it for 5 mins. Repeat." elementName="Wash coverslips in detergent" substepsCollapsed="false"/></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"/><FixedStep description="Block with 1% NDS/Abdil (spin before use) for 30-60 mins." elementName="Block with Abdil" substepsCollapsed="false"/><FixedStep 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" substepsCollapsed="false"/><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><FixedStep elementName="Channels" substepsCollapsed="false"><FixedStep backgroundColour="0:0:255" elementName="Blue Channel" substepsCollapsed="true"><TextField elementName="Channel Name / Antibody" substepsCollapsed="false" value="DAPI"/><TextField default="1:500" description="Dilute in Abdil.&#10;Incubate on cells for 1 hour." elementName="Primary Antibody Dilution" substepsCollapsed="false" value="1:500"/><TextField default="488" elementName="Seconday Antibody" substepsCollapsed="false"/></FixedStep><FixedStep backgroundColour="0:255:0" elementName="Green Channel" substepsCollapsed="true"><TextField elementName="Channel Name / Antibody" substepsCollapsed="false" value="Anti-Aurora-B"/><TextField default="1:500" description="Dilute in Abdil.&#10;Incubate on cells for 1 hour." elementName="Primary Antibody Dilution" substepsCollapsed="false" value="1:500"/><TextField default="488" description="Usually diluted 1:150.&#10;Usually use Alexa dyes. Green is 488." elementName="Seconday Antibody" substepsCollapsed="false" value="Anti-Rabbit 488"/></FixedStep><FixedStep backgroundColour="255:0:0" elementName="Red Channel" substepsCollapsed="true"><TextField elementName="Channel Name / Antibody" substepsCollapsed="false" value="DM1-alpha"/><TextField default="1:500" description="Dilute in Abdil.&#10;Incubate on cells for 1 hour." elementName="Primary Antibody Dilution" substepsCollapsed="false" value="1:500"/><TextField default="568" description="Usually diluted 1:150.&#10;Usually use Alexa dyes." elementName="Secondary Antibody" substepsCollapsed="false" value="Anti-Mouse 568"/></FixedStep><FixedStep elementName="Far Red Channel" substepsCollapsed="false"><TextField elementName="Channel Name/ Antibody" substepsCollapsed="false" value="ACA"/><TextField default="1:500" description="Dilute in Abdil.&#10;Incubate on cells for 1 hour." elementName="Primary Antibody Dilution" substepsCollapsed="false" value="1:500"/><TextField default="Cy5" description="Usually diluted 1:150.&#10;Usually use Alexa dyes." elementName="Secondary Antibody" substepsCollapsed="false" value="Anti-Human Cy5"/></FixedStep></FixedStep></ProtocolTitle><FixedStep elementName="&#10;      Observations&#10;    &#10;  " substepsCollapsed="false"><ObservationDefinition backgroundColour="255:153:153" elementName="&#10;      Time of Anaphase onset&#10;    &#10;  " observationAttributeTermID="PATO:0001309   time" observationEntityTermID="GO:0007091   mitotic metaphase/anaphase transition" observationType="Number" observationUnitsTermID="UO:0000031   minute" substepsCollapsed="false"/><ObservationDefinition backgroundColour="204:153:51" description="kjh" elementName="&#10;      Metaphase alignment&#10;    &#10;  "/></FixedStep><FixedStep description="&#10;      These phenotypes are not mutally exclusive, except &amp;quot;Alignment OK&amp;quot;, which &#10;      means that none of the other defects occured. &#10;    &#10;  " elementName="&#10;      Metaphase alignment at Anaphase entry&#10;    &#10;  " substepsCollapsed="false"><TextField elementName="&#10;      Complex Phenotype. Name:&#10;    &#10;  " substepsCollapsed="true" value="Alignment OK"><OntologyLookupServiceField elementName="&#10;  " ontolgoyTermId="GO:0007080   mitotic metaphase plate congression" ontologyId="GO" ontologyTermName="mitotic metaphase plate congression" substepsCollapsed="false"/><OntologyLookupServiceField elementName="&#10;  " ontolgoyTermId="PATO:0000461   normal" ontologyId="PATO" ontologyTermName="normal" substepsCollapsed="false"/><OntologyLookupServiceField elementName="&#10;  " ontolgoyTermId="PATO:0000168   relative time" ontologyId="PATO" ontologyTermName="relative time" substepsCollapsed="false"/><OntologyLookupServiceField elementName="&#10;  " ontolgoyTermId="GO:0007091   mitotic metaphase/anaphase transition" ontologyId="GO" ontologyTermName="mitotic metaphase/anaphase transition" substepsCollapsed="false"/></TextField><TextField elementName="&#10;      Complex Phenotype. Name:&#10;    &#10;  " substepsCollapsed="false" value="Multipolar spindle"><OntologyLookupServiceField elementName="&#10;  " ontolgoyTermId="GO:0031616   spindle pole centrosome" ontologyId="GO" ontologyTermName="mitotic metaphase plate congression" substepsCollapsed="false"/><OntologyLookupServiceField elementName="&#10;  " ontolgoyTermId="PATO:0000650   supernumerary" ontologyId="PATO" ontologyTermName="having supernumerary physical parts" substepsCollapsed="false"/></TextField><TextField elementName="&#10;      Complex Phenotype. Name:&#10;    &#10;  " substepsCollapsed="false" value="No chromosome alignment"><OntologyLookupServiceField elementName="&#10;  " ontolgoyTermId="GO:0007080   mitotic metaphase plate congression" ontologyId="GO" ontologyTermName="mitotic metaphase plate congression" substepsCollapsed="false"/><OntologyLookupServiceField elementName="&#10;  " ontolgoyTermId="PATO:0000462   absent" ontologyId="PATO" ontologyTermName="absent" substepsCollapsed="false"/><OntologyLookupServiceField elementName="&#10;  " ontolgoyTermId="PATO:0000713   relative time value" ontologyId="PATO" ontologyTermName="relative time" substepsCollapsed="false"/><OntologyLookupServiceField ontolgoyTermId="GO:0007080   mitotic metaphase plate congression"/></TextField></FixedStep></ProtocolTitle>