http://www.protocol-online.org/forums Thu, 02 Feb 2012 19:51:50 +0000 3600 http://www.protocol-online.org/forums/topic/24261-a-query-regarding-post-fixation-for-immunofluorescence/
I am trying to measure c-Fos expression in free floating brain sections using immunofluorescence. For some reason, I don't see the c-Fos in the nucleus. I see it mostly in the cytosol and dendrites. In addition, the signal to noise ratio is pretty bad..

I have managed to focus the problem on the post-fixation process; I have asked a colleague to use her brain sections. Using my staining protocol and same antibody I was able to view an amazing signal, located in the nucleus and with a really good signal to noise ratio.

There are some differences between out post-fixation protocols.
The protocol I use for post-fixation is:
96 hours in 0.01M PBS + 30% sucrose + 1% PFA in 4C.
after the brain has sinked I take it out and freeze the brain in -80C
couple of days \\ weeks later - I defreeze it and use the microtom to slice it.

Her post fixation is different than mine. First she keeps the brain in 0.01M PBS + 4% PFA over night, following by a gradient of PBS + sucrose (10% for 24 hours and then 20% for 24 more hours). She does not freeze the brain after the post fixation process, but keeps it in 4C until slicing.


What do you think causes my problem? Which protocol do you use?

Thanks in advance!
Karen.]]> Thu, 02 Feb 2012 19:51:50 +0000 http://www.protocol-online.org/forums/topic/24261-a-query-regarding-post-fixation-for-immunofluorescence/ http://www.protocol-online.org/forums/topic/24005-gfapng2-flow-cytometry/
Does anyone know of any good conjugated antibodies for GFAP and NG2 which can be used for flow cytometry? I managed to find some conjugated anti-GFAP in millipore but it's for human and I'm working on mice.

Any help appreciated!]]> Thu, 12 Jan 2012 17:10:51 +0000 http://www.protocol-online.org/forums/topic/24005-gfapng2-flow-cytometry/ http://www.protocol-online.org/forums/topic/23988-e-phys-improving-10-90-rise-time/ Wed, 11 Jan 2012 16:57:40 +0000 http://www.protocol-online.org/forums/topic/23988-e-phys-improving-10-90-rise-time/ http://www.protocol-online.org/forums/topic/23409-sh-sy5y-differentiation-protocolsquestions/
I'm fairly new to cell culture and work with SH-SY5Y neuroblastoma cells. I need to differentiate them, and plan on using a final concentration of 10 uM Retinoic Acid. However, I can't seem to find a consensus in the literature about what media to differentiate in. I normally grow them in 1:1 DMEM:Ham's F12 + 10% FBS and Antibiotics. I have heard mostly 2 schools of thought, using complete medium + RA or using medium with 1% FBS + RA.

What would you suggest? Are there pros/cons to each? Will moving my cells from 10% FBS to 1% shock them?

Thank you!]]> Mon, 14 Nov 2011 14:20:13 +0000 http://www.protocol-online.org/forums/topic/23409-sh-sy5y-differentiation-protocolsquestions/ http://www.protocol-online.org/forums/topic/23399-beta-amyloid-25-35-preparationhelp-needed/ hi...is it important to prepare the aggregate form of beta amyloid (25-35) in a serum free media (DMEM or RPMI). what if i prepare it in serum containing DMEM for my treatment? Does the serum may interfere with the beta amyloid (25-35)? Sun, 13 Nov 2011 16:43:14 +0000 http://www.protocol-online.org/forums/topic/23399-beta-amyloid-25-35-preparationhelp-needed/ http://www.protocol-online.org/forums/topic/23379-astrocyte-specific-proteins/
I am working on astrocytes in mouse models, and I'm interested in studying the inflammatory response in CNS.

Astrocytes are known to have a role in inflammatory response, though microglia have been reported to be more prominent.

However, I am curious to know about growth factors or soluble proteins released specifically by astrocytes.

If there is anyone working in this area, I would be happy to receive some comments or ideas on astrocyte specific proteins, other than GFAP and s100beta.

all suggestions in this regard are welcome.

Thanx for any help.]]> Thu, 10 Nov 2011 08:05:07 +0000 http://www.protocol-online.org/forums/topic/23379-astrocyte-specific-proteins/ http://www.protocol-online.org/forums/topic/23324-analysis-of-different-results-on-anxiety-related-behavior-on-the-same-group/ IF someone is familiar with behavioral testing on mice. I did elevated plus maze and open field test on mice in order to study anxiety behavior. I have 2 animal groups. In elevated plus maze, I could see difference between the 2 groups . as for the open field, the difference isnt statistically significative.
I dont really know how to interpret those results
thanks for help]]> Thu, 03 Nov 2011 23:01:37 +0000 http://www.protocol-online.org/forums/topic/23324-analysis-of-different-results-on-anxiety-related-behavior-on-the-same-group/ http://www.protocol-online.org/forums/topic/23226-mouse-max-discernment-btw-colors/
If you were to design a behavioral mouse experiment in which:


Mouse sees Color A -> do behavior A

Mouse sees Color B -> do behavior B


Which 2 colors would you choose (A and B ), so that the mouse can easily discern between the two colors.


Let me make it easy, your choices are Red, Green, Amber, or Yellow.


Thx.]]> Wed, 26 Oct 2011 14:30:20 +0000 http://www.protocol-online.org/forums/topic/23226-mouse-max-discernment-btw-colors/ http://www.protocol-online.org/forums/topic/23213-primary-cell-isolation-from-cns-how-to-remove-myeline-debris/
I have a question for you,
I just started to do some culture of primary cell from mouse CNS,
but after dissociation/isolation, myeline debris is all over and make it almost impossible for me to count the # of cells.

Does anyone know some easy (and hopefully inexpensive way) to remove the debris?

I know there is myeline removal microbeads in market, but I prefer some other method since our lab does not use the microbeads system.

I prefer some density gradient protocol, so if anyone have any suggestion, please let me know.

Thanks in advance]]> Tue, 25 Oct 2011 18:21:51 +0000 http://www.protocol-online.org/forums/topic/23213-primary-cell-isolation-from-cns-how-to-remove-myeline-debris/ http://www.protocol-online.org/forums/topic/23159-labtutor-version-40/
Could anyone advice on how we could save the data and open it later for analysis?

Note: The analysis hasn't been done. Is there a way to save the data or the data is saved?]]> Thu, 20 Oct 2011 14:03:19 +0000 http://www.protocol-online.org/forums/topic/23159-labtutor-version-40/ http://www.protocol-online.org/forums/topic/22944-how-to-determine-apoptotic-cell-types-in-vivo/
I am working on a Neuro-degenerative mouse model. We used Tunel staining to show the apoptotic level in brain of these mice. However, i was asked to determine the cell types were involved into these apoptotic events. The apoptotic study using tunel were performed on paraffin sections.

So, any advice?

Thanks]]> Thu, 29 Sep 2011 20:01:13 +0000 http://www.protocol-online.org/forums/topic/22944-how-to-determine-apoptotic-cell-types-in-vivo/ http://www.protocol-online.org/forums/topic/22846-drosophila-embryo-collection/
Thanks!!]]> Mon, 19 Sep 2011 21:29:32 +0000 http://www.protocol-online.org/forums/topic/22846-drosophila-embryo-collection/ http://www.protocol-online.org/forums/topic/22797-gfpautofluorescence-in-wild-type-mouse-brain/
I have a transgenic line of mice which express my protein of interest as well as a GFP reporter. I'm taking fixed frozen sections from these mice and trying to localize expression of the vector with fluorescent microscopy. However, I'm seeing what looks like GFP positive cells in the (negative control) wild-type mouse as well. I've repeated it with a couple mice so I'm confident it's not just a matter of grabbing the wrong mouse.

The cells appear to be relatively large and in the midbrain/brain stem areas. Has anyone experienced this before?

Unfortunately my GFP expression in the transgenic mice is seemingly low as well (and in similar areas to where I'm seeing it in the WT) so I'm not sure if I'm simply seeing normal "background" levels of fluorescence or something else. The vector is pIRES from Clontech.

Thanks]]> Wed, 14 Sep 2011 22:02:32 +0000 http://www.protocol-online.org/forums/topic/22797-gfpautofluorescence-in-wild-type-mouse-brain/ http://www.protocol-online.org/forums/topic/22571-brdu-in-lesioned-brain/
I'm trying to set-up BrDU IHC in lesioned brain and I found that it is surprisingly not working. Everything goes perfect for the age-matched controls, that had the same perfusion, storage, cutting and IHC. But on my lesioned samples I see no +cells at all, which is quite weird as I already characterized them by PCNA, ki67, phh3, etc.
Has anyone find any similar problem? I'm trying not to change things in the protocol, as different cycles with boiling citrate or different T with HCl...

Thanks!!]]> Mon, 22 Aug 2011 15:28:07 +0000 http://www.protocol-online.org/forums/topic/22571-brdu-in-lesioned-brain/ http://www.protocol-online.org/forums/topic/22535-drosophila-anatomy-of-the-brain/ I am new to studying the drososphila and was wondering if any one knows of a book that i can read to get a basic understanding of the fly brain anatomy Wed, 17 Aug 2011 17:12:47 +0000 http://www.protocol-online.org/forums/topic/22535-drosophila-anatomy-of-the-brain/ http://www.protocol-online.org/forums/topic/22476-drosophila-tracking/ I am working with the fly and was wondering if there is a software to track the fly that i can download and use with the videos that i have. Fri, 12 Aug 2011 15:54:37 +0000 http://www.protocol-online.org/forums/topic/22476-drosophila-tracking/ http://www.protocol-online.org/forums/topic/22426-ngf-toxicity-and-pc12-degeneration/

On the first trial, I treat my dear PC12 with 100ng/ml NGF but it's not working (maybe because of the 'not fully dissolved NGF'). So I increase the concentration for the next trial. The neurite start to grow but on the 5th and 6th day, my dear cell start to degenerate . Does high concentration of NGF might be the cause of the degeneration PC12? Is it toxic to the cell?]]> Tue, 09 Aug 2011 03:03:04 +0000 http://www.protocol-online.org/forums/topic/22426-ngf-toxicity-and-pc12-degeneration/ http://www.protocol-online.org/forums/topic/22403-pyramidal-dendrites-or-multipolar-which-is-more-important-for-morphometric-anal/ I am studying the morphological structure of mouse neurons. I would like to get some suggestions on which is the best criteria for morphological analysis for mouse neurons. I have seen that pyramidal neurons are more widely studied in papers than multipolar or bi-polar. Why is this so? Is there a standard reference paper that I can follow?
Please give me some suggestions in this regard. Thanx in advance]]> Sat, 06 Aug 2011 03:13:41 +0000 http://www.protocol-online.org/forums/topic/22403-pyramidal-dendrites-or-multipolar-which-is-more-important-for-morphometric-anal/ http://www.protocol-online.org/forums/topic/22392-svz-culture/ after i dissect SVZ, trypsinize and add medium, i observe that
medium culture became pink and turbid. next day, the white mucus appear.
I have this problem everytime. I think it might be comtamination but i cannnot observe
the foreign body under microscope.
after i passage this group of cell, cells decrease growth rate and death.
- my medium contain pen/strep, i might be the cause the i cannot observe the contamination?
Can anyone help me to cure this problem? Please!!!]]> Fri, 05 Aug 2011 06:18:54 +0000 http://www.protocol-online.org/forums/topic/22392-svz-culture/ http://www.protocol-online.org/forums/topic/22298-viral-transfection-to-neurosphere-in-vitro/
Does anyone ever did similar experiment? Anything that she needs to be careful about like selection of virus and such?
or any paper to read before design virus?

Any suggestion helps

Thanks in advance]]> Wed, 27 Jul 2011 18:07:30 +0000 http://www.protocol-online.org/forums/topic/22298-viral-transfection-to-neurosphere-in-vitro/