Author Topic: Storing cut rat brain @ -80 before immunostaining  (Read 2807 times)

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Offline UniCorn24

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Storing cut rat brain @ -80 before immunostaining
« on: March 21, 2011, 01:41:15 PM »
Hi there.

I snap freeze fresh rat brains covered in OCT,  in isopentane cooled in liquid Nitrogen. I generally store these at -80 until sectioning.

after sectioning I put my cut slides back into the -80, until I do immunofluorescence protocol. I used to perfuse fix the brain with 4% PFA and cryoprotect with 20% sucrose, but due to loss of antigenicity I switched to a fresh frozen approach.

Has anyone experienced any problems with this?, I am worried as I don't fix my slides (often with Methanol/Acetone) until I do my immunoprotocol.

If anyone has any advice or tips they would be greatly appreciated.

thanks

Storing cut rat brain @ -80 before immunostaining
« on: March 21, 2011, 01:41:15 PM »

Offline CanuckPhD

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Re: Storing cut rat brain @ -80 before immunostaining
« Reply #1 on: March 22, 2011, 08:38:03 AM »
The length of time that a cut unfixed slide can be stored in the -80 depends on the antigen being examined. I usually try to perform all of the required staining within 3 weeks after cutting and the results have been consistent. However, I examine the usually microglia markers (CD11b, Iba-1, CD68, MHC II etc), astrocytes (GFAP) and myelin (MBP, Nogo, NG2 etc). Your antigen and antibody might be more sensitive to the drying artefact that is found in the -80. Using one of the machines that can vacuum pack slides might be a good idea.

Offline UniCorn24

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Re: Storing cut rat brain @ -80 before immunostaining
« Reply #2 on: July 02, 2011, 12:26:17 PM »
That's great, thanks very much for the reply...I have great results with some antigens and others no results!!! such is life!!

Offline pandanac

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Re: Storing cut rat brain @ -80 before immunostaining
« Reply #3 on: July 06, 2011, 04:13:23 AM »
I have been having a strange problem of late...  I perfuse the rat with 4% PFA and store the brain in 4% pfa (post fixation) 1 day, and then transfer it to 30% sucrose till it sinks. I remove it and embedd in OCT and freeze in -80. However I use a mold (from Polysciences) to put my brain in OCT. I had sectioned slices of 20 micron thickness. I am seeing holes in the brain slices. I dont know where the problem lies? The perfusion was very good. The brain sank down after a day or 2 and only after making sure that the brain sank down in sucrose, i had put it in OCT and sectioned it the next day...This is a new problem.. that i am having... earlier I used to use a chewing gum wrap (the one we get with Orbit chewing gum..sounds crude... but works well).. now when I am using this mold (reason is that I need to cut 80% of the brain and the chewing gum wrap is small in size)...

Please could anyone help me in this.. few crucial brains.. have been messed up and I cant afford to do it any further... plzzzzzzz
plzzzzzz help mee.

Re: Storing cut rat brain @ -80 before immunostaining
« Reply #3 on: July 06, 2011, 04:13:23 AM »