Hi all,
I need some advice for mounting brain tissue sections (50um) for immunofluorescenct detection. At the moment I plan to dual stain with two different primary antibodies and then mount in DAPI medium. I also have couple of questions:
1) Should I leave my sections to dry before mounting or mount immediately after the final wash steps? I've seen that some people air dry while some mount while the tissue is still wet.
2) I have a large number of sections to mount so was wondering how long can I keep the tissue in PBS before mounting - can I store in the fridge out of the light for an extensive amount of time?
Thanks in advance!
David
Hello
Look at links here will help you
Good Luck
http://penlabs.com/wp-content/uploads/2014/03/IFK-PROTOCOL_050814_PLI-Inc1.pdf
http://www.cbi.pitt.edu/protocols/tissueimuno.htm
http://www.bio-protocol.org/fllist.aspx?fl3=533&weekcount=6
https://www.peprotech.com/en-GB/Pages/ProtocolItem/Immunofluorescence(c)_General_Animal_Brain_Tissue
https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-paraffin-embedded-tissue
http://www.nature.com/nprot/journal/v1/n4/full/nprot.2006.265.html
Hi David. For mounting, you can do either way. I usually position my slide vertically to allow fluid to fall down naturally for ~5mins. I don't really allow it to dry entirely. I mount the slides with Dako fluorescence mounting medium and view under a microscope immediately. Works fine with me. My colleagues sealed the coverslip with nail polish in addition to the mounting step, and wait ~1hour to allow the nail polish to dry before viewing under the microscope.
About storing in PBS for a long time, I won't recommend that actually. In my faculty, the secondary antibodies are very lacking, that I won't risk wasting my antibodies. Maybe you can opt to section and stain some of your embedded tissue one by one, and keep the other embedded tissues (the ones that you can do later) at 4°C. Storing embedded tissues at 4°C can last maximum of 1 year, but don't store longer than that because antibodies may not be able to detect your target proteins if stored too long. All the best!
Hi David,
1) You do not need to dry your sections after immunohistochemistry, prior to or after mounting them. Put a drop of mounting medium on a glass slide, take your section with a brush form PBS and place it on the medium and then place a cover slip on top.
2) You can keep your sections in PBS for several days in fridge, before mounting. If you need to keep your sections in PBS before mounting for a longer time, you will need to change your PBS every couple of days and need to work sterile, and you can store in the fridge out of the light for a weeks.
For further hints on immunological methodology check out my previous answers, I am sure you will find useful info.
best wishes, Refik
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