[Histonet] EGFP expression in mouse testis cryosections

[Anatoli Gleiberman]

Hi Muralidhar,

Fix your samples in 4% formaldehyde on PBS (you can prepare it from powder or from 20% solution in water sold by Electron Microscopy Sciences) about 4h at room temp, wash with PBS (at this step you can store your samples at +4 C for several days or even weeks, cryoprotect by incubation in sucrose/PBS solution (10% sucrose until samples sink - usually overnight at +4 C, 30% sucrose until sinking - also usually overnight), transfer into cryomold, fill it with cryo-embedding medium (O.C.T. or other compounds - we are using Neg-50 from Richard-Allan)freeze (you can freeze them fast in mixture or isopentane/dry ice or slow in -80 C freezer - does not matter) and make cryosections. Good GFP expression is well preserved in formaldehyde-fixed material. If fluorescence is low or invisible for some reasons you can stain these sections with either rabbit or chicken anti-GFP antibody from Abcam or chicken anti-GFP antibody from Aves Lab (all of them are good, work in dilutions 1:500 and more) and secondary either AlexaFluor488 or DyLight488 conjugated, up to you. Alternatively, you can embed your samples in paraffin using routine protocol. In this case your native eGFP fluorescence will be lost completely, but you can still visualize eGFP with antibodies mentioned above following heat antigen retrieval - and use either fluorescence or peroxidase technique. I don't remember how high is testis autofluorescence - maybe peroxidase will be better in your case. For brain, intestine and even liver I usually use fluorescence.

Good luck.

Anatoli Gleiberman, PhD
Director of Histopathology
Cleveland Biolabs, Inc
73 High Street
Buffalo, NY 14203
phone:716-849-6810 ext.354
fax:716-849-6817
e-mail: AGleiberman <@t> cbiolabs.com

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of murali
Sent: Thursday, March 03, 2011 4:07 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] EGFP expression in mouse testis cryosections

dear all,

i am relatively new to histochemistry. i would like to visualize EGFP
expression mouse testis. i am not sure if this is expressed in cytoplasm or
nucleus.  what kind of staining i should follow and are there some protocols
that can be of help for me. please let me know if i need to provide more
details about my experiment.

thanks
regards
murali

-- 
Muralidhar Metta
Post Doctoral Fellow
Unit of Animal Genomics
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Belgium
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