Editing
Daniel:Notebook/RNAFACS/2015-10-7
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
=RNA Scope Trial Run= [[Daniel:Notebook/RNAFACS|Back to Calendar]] Now that I know that the nuclear adhesion protocol works its time to test out RNA Scope (fluorescent), including trying out the Huang lab microscope. ==Nuclei Adhesion== #Clean glass slide in 100% EtOH; allow to air dry ##Draw a square around the desired adhesion area using a hydrophobic barrier pen #Remove cells from -80C ##Separate into two tubes (15 uL ea) and spin down for 5 min at 1000g ##Remove supernatant, careful not to disturb the pellet #Add 500uL of 4% paraformaldehyde to the tube and wait '''5 min''' #Centrifuge cells for 5 min at 1000g ##Remove supernatant, careful not to disturb the pellet #Add 500 uL of 70% EtOH #Centrifuge cells for 5 min at 1000g ##Remove supernatant, careful not to disturb the pellet #Resuspend nuclei in 20 uL 70% EtOH #Deposit on to glass slide #Use a coverslip to gently spread the 20 uL across the slide ##Be careful not to scratch the slide #Dry the slide using a heating block at 60C for 10-15 min ==RNA Scope== '''Sample Matrix''' {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:12pt" | align="center" width="65" height="15" valign="bottom" | |style="background-color:#CCC0DA;font-weight:bold" width="94" align="center" valign="bottom" | Slide 1 |style="background-color:#CCC0DA;font-weight:bold" width="65" align="center" valign="bottom" | Slide 2 |- style="font-size:12pt" | height="15" valign="bottom" | C1 | valign="bottom" | RBFOX3/NeuN | align="center" valign="bottom" | +Control |- style="background-color:#D9D9D9;font-size:12pt" | height="15" valign="bottom" | C2 | valign="bottom" | -Control | align="center" valign="bottom" | +Control |- style="font-size:12pt" | height="15" valign="bottom" | C3 | valign="bottom" | FEZF2 | align="center" valign="bottom" | +Control |} ===Nuclei Prep=== #Rehydration ##Incubate slides in 70% EtOH for 2 minutes ##Incubate slides in 50% EtOH for 2 minutes ##Incubate slides in 1X PBS for 10 minutes ##May want to retrace hydrophobic barrier; can skip #Apply Pretreat 3 ##Remove excess liquid; add 2-4 drops of pretreat 3 ##Incubate for 10 min at RT (in oven) ##Flick to remove excess liquid ##Rinse in fresh 1X PBS; be sure to agitate to clean well ===Fluorescent Assay=== #Prepare-Reagents ##Prepare probes by warming to 40C for 10 minutes, then cool to RT ##Briefly spin down C2 probes to collect the liquid ##Combine C2 and C1 probes by pipetting into a new 1.5mL tube at 1:50 ratio ##Prepare 1X Wash Buffer ##Place AMP1-4 FL reagents at RT ##Ensure HybEZ OVEN and Control Tray are at 40C #Hybridize Probe ##Flick to remove excess liquid; add 2-4 drops of probe ##Incubate for 2 hrs at 40C ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes #AMP1-FL ##Flick to remove excess liquid ##Add 2-4 drops of '''AMP1-FL'''; Incubate for 30 minutes at 40C ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes #AMP2-FL ##Flick to remove excess liquid ##Add 2-4 drops of '''AMP2-FL'''; Incubate for 15 minutes at 40C ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes #AMP3-FL ##Flick to remove excess liquid ##Add 2-4 drops of '''AMP3-FL'''; Incubate for 30 minutes at 40C ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes #AMP4-FL ##Flick to remove excess liquid ##Add 2-4 drops of '''AMP4-FL ALT B'''; Incubate for 15 minutes at 40C ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes ##Flick to remove excess liquid; submerge in 1X Wash Buffer; incubate 2 minutes #Counterstain and Mount ##Flick to remove excess liquid; ##Add 2-4 drops '''DAPI'''; Incubate 30 sec at RT ##Remove DAPI and immediate place 1-2 drops of fluorescent mounting medium ##Seal with a coverslip; avoid bubbles #Store in the dark at 4C #Recommended Viewing at 20-40X magnification
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information