I am new in this field and I have a question.
I work with adeno virus with GFP marker.
what is the best protocol for cryosections in order to see well the GFP expression ?
Thank you for your answer. but in this article they used parffin and i workwuth cryosection.
im injected adeno virus into rat heart and after a week did cryosection.
the problem is that i cant observe any GFP in the tissue.
I didnt fix the tissue before rmbedding in OCT, and i dont know if it is a problem,
i know that some people use PFA and sucrose for fixation, and i wanted to know what is the protocol for this?
Please do refer to the Immuno Protocol at http://www.scientistsolutions.com/a7161-protocol-immuno+protocol.aspx
The protocol is not ok for what you want i would suggest a few modifications.
Your cutting staining protocol is controlled by how you would analyse it. I would suggest stereoligical quantification.
1)Perfuse animal with PBS (mouse 30 ml, rat 100ml)
2) 30 ml 4% paraformaldehyde 4ºC, block the brain, in vivo post fix for two hours at room temperature (r.t.)
3) allow the brain to sink in 30% sucrose in PB for 48 hours. Snap freeze. then either cyrosection onto slides 20- 30 micron or cut for free floating 30-50 micron. Never let the tissue you are sectioning touch the oct.
4) wash sections in 0.1M Phosphate Buffered Saline (PBS) (pH 7.4) with 1% TritonX100 (3x 5 mins)
5) with GFP never use unmasking (hcl or citate)
6) block wiht 0.1M PBS (pH 7.4) + 1% TritonX100 + 5% normal goat serum if secondary is raised in goat or sheep(1hr i)
7) prior to incubating overnight (4ºC) with anti-GFP in the same solution but 1% ngs
8) Wash 3x then anti GFP secondary for 3 hours. Very impt if you want to quatify.3x wash
9) use an antifade mounting media
thank you !
I have another question that.
why should I use antibodies for GFP and cant look at the GFP itself ?
If your adeno virus transfection in good and high you may not need to use it. Denfinately look at the sections before you immunostain them.
However, I think you will need to as:
1) it will amplify the signal enableing you to see many more transfected neurones.
2) GFP tends to fade, The alexa fluor type flurophores are very resistant to fading and are very bright.
3) if you use a HRP secondary you can use it without fluresence,