http://www.protocol-online.org/forums/index.php Fri, 20 Nov 2009 17:15:12 -0800 3600 http://www.protocol-online.org/forums/index.php?showtopic=11472
I appreciate this. Cheers.]]> Wed, 11 Nov 2009 12:25:52 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11472 http://www.protocol-online.org/forums/index.php?showtopic=11438 what is the significance of finding out the ratio of EPSC2:EPSC1? As I know it is used to determine the change in probability of transmitter release. But why so? Tue, 10 Nov 2009 02:41:59 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11438 http://www.protocol-online.org/forums/index.php?showtopic=11429 ]]> Mon, 09 Nov 2009 11:24:09 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11429 http://www.protocol-online.org/forums/index.php?showtopic=11315
I have no experience in electron microscopy. If I have PFA-fixed brain tissues, can I use them for electron microscopy?
In the literature, I found that PFA+glutaraldehyde fixation is needed, can this glutaraldehyde fixation be given later to the already PFA fixated tissue? What is the reason of using different concentrations (2%PFA/2%glutaraldehyde, or 4%PFA/0,1%glutaraldehyde)? What does the glutaraldehide to the sample?]]> Tue, 03 Nov 2009 05:38:51 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11315 http://www.protocol-online.org/forums/index.php?showtopic=11050 Thu, 22 Oct 2009 13:30:36 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11050 http://www.protocol-online.org/forums/index.php?showtopic=10398
I got some problems using the criostat (10 micron) in MCAO brains, my impression is that the tissue after stroke induction become fragile and this make me difficult to get good slices .
I looking for an alternative protocol able to get me thick slices to treat with anti-bodyes.

thanks

tizzy]]> Mon, 21 Sep 2009 03:49:33 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10398 http://www.protocol-online.org/forums/index.php?showtopic=9161
I am trying to staing mouse brain sections for sox-2 (frozen, FIXED WITH PFA 4%).
The problem is that I get a lot of positive stainig - not only in the DG and SVZ, but also all over the brain.
It doesnt look like backgroung. Did anyone tryied to use sox-2 antibody ? do you expect to see so many positive cells?

Thanks,
Rona ]]> Wed, 15 Jul 2009 23:14:54 -0700 http://www.protocol-online.org/forums/index.php?showtopic=9161 http://www.protocol-online.org/forums/index.php?showtopic=8966
The classical Golgi impregnation method is very elegant in its simplicity:
1. Immerse a block (approx. 10x5 mm) of formol-fixed (or paraformaldehyde- glutaraldehyde-pefused) brain tissue into a 2% aqueous solution of potassium dichromate for 2 days
2. Dry the block shortly with filter paper.
3. Immerse the block into a 2% aqueous solution of silver nitrate for another 2 days.
4. Cut sections approx. 20-100 µm thick.
5. Dehydrate quickly in ethanol, clear and mount (e.g., into Depex).

thanks!]]> Sat, 04 Jul 2009 21:06:13 -0700 http://www.protocol-online.org/forums/index.php?showtopic=8966 http://www.protocol-online.org/forums/index.php?showtopic=8684 The problem is after I seed cells onto laminin or fibronectin-coated plates and leave them overnight in the incubator, cells are clumpy the following day.. obviously they don't attach to the plates but they tend to form clusters. On the fifth day, most clusters attach to the plates and elongate processes. This can be seen in laminin and fibronectin-coated plates , but not neuronal cultures in polylysine-coated ones.

Recently I have found this article 'Phosphothioated Oligodeoxynucleotides Induce Nonspecific Effects on Neuronal Cell Adhesion in a Growth Substrate-Dependent Manner'
Briefly, they added CpG (oligonucleotides, TLR9 ligand) to neuronal culture in Polylysine or laminin-coated plates and cell detachment happened in laminin-coated but no Polylysine-coated plates. The photos in this article look exactly like my cultures so I make me think that there may be bacterial DNA (esp. E.coli DNA which is also TLR9 ligand) contamination in something that I have added to cells.

I am thinking also about my plate-coating method may be wrong. I dilute laminin and fibronectin in PBS with Ca++ and Mg++ and add onto the plates, incubate 4'c overnight, block with BSA and wash with PBS without CA and Mg. I have tried coating laminin on top of polylysine but I doesn't solve the problem

Any suggestions of what's happening to my cultures? or source of bacterial DNA contamination?

ps. I am using Neurobasal, B27, PDS, penicillin/streptomycin and glutamine medium// trypsin, DNase and FCS for cell dissociation.]]> Thu, 18 Jun 2009 03:02:43 -0700 http://www.protocol-online.org/forums/index.php?showtopic=8684 http://www.protocol-online.org/forums/index.php?showtopic=8627 I am starting my new project in my new lab about spinal cord injury model using mouse.

Unfortunately, there is no one in our lab who has experience in this injury model although our lab is neuro/developmental bio lab.

So, does anyone know good HP or paper for me to read to understand the basic procedures or info to establish this model?
or
any good methods to analyze the spinal cord (site of injury) after the experiment

Any suggestion helps

Thank you in advance]]> Mon, 15 Jun 2009 23:27:38 -0700 http://www.protocol-online.org/forums/index.php?showtopic=8627 http://www.protocol-online.org/forums/index.php?showtopic=8421 Does anyone who has experience in handling of primary neuron of fly could provide me detail protocol? I also wonder what reagent you're using to digest the brains? What I'm using now is collagenase but it seems not very powerful...

thanks in advance...]]> Thu, 04 Jun 2009 01:18:23 -0700 http://www.protocol-online.org/forums/index.php?showtopic=8421 http://www.protocol-online.org/forums/index.php?showtopic=8321 We are having trouble finding another that works well. Please help!!]]> Fri, 29 May 2009 10:00:20 -0700 http://www.protocol-online.org/forums/index.php?showtopic=8321 http://www.protocol-online.org/forums/index.php?showtopic=8296 Hi, could anyone help me out with information on how to culture the mouse neuroblastoma cell line Neuro 2A and rat neuroblastoma line B104 ? I could not figure out whether a particular coating is needed for those cells. And, does somebody have experience in how those cells grow in RPMI Medium + 10%FCS (instead of the published DMEM +10%FCS)? Thanks. Thu, 28 May 2009 07:39:16 -0700 http://www.protocol-online.org/forums/index.php?showtopic=8296 http://www.protocol-online.org/forums/index.php?showtopic=8059 Hello,

I am interested in neurodevelopment and I am planning an experiment about it but I couldn't decide the appropiate cell type. Which cell type is more proper for studying expression profile of neurodevelopmental genes?

PC12 (pheochromocytoma of rat adrenal medulla) or P19 (murine embrional carcinoma stem cell line)?

I would be glad if anyone can help me.

Thank you]]> Thu, 14 May 2009 01:04:38 -0700 http://www.protocol-online.org/forums/index.php?showtopic=8059 http://www.protocol-online.org/forums/index.php?showtopic=7606
I have few problems regarding measurement of calcium influx in neurosynaptosomes. I am making the preperation by the method desribed in book"neurotransmitter methods", which involves a two stage filteration of brain homogenate , firstly with 100micron filter and again with 8 micron filter, the resulting suspension is belived to be containing neurosynaptosomes. Next the preperation is loaded with Fura2 AM and the resulting fluroscence measured.I need to know whether there is any method to check the viability of the neurosynaptosomes preperation as I`m not getting the desired activity. Is there any other method to prepare neurosynaptosomes.

It would be very kind to anyone of u who can help me in this regard.

Thank you]]> Sun, 19 Apr 2009 22:44:48 -0700 http://www.protocol-online.org/forums/index.php?showtopic=7606 http://www.protocol-online.org/forums/index.php?showtopic=7296
I am trying to do a Brdu staining on Spinal cord sections that have GFP cells, the purpose is to see if those cells have been proliferating because of a disease or not. since I have very few good sections( they are PFA perfused, frozen sections), I am afraid I might loose GFP because of harsh treatment that involves Brdu staining, does any one has any suggestion?]]> Wed, 01 Apr 2009 16:37:47 -0700 http://www.protocol-online.org/forums/index.php?showtopic=7296 http://www.protocol-online.org/forums/index.php?showtopic=7280 Basicly that is my question, is kainic acid from Nanocs USA any good ? Wed, 01 Apr 2009 02:42:24 -0700 http://www.protocol-online.org/forums/index.php?showtopic=7280 http://www.protocol-online.org/forums/index.php?showtopic=7104
Does anyone here knows a good retroviral vector (moloney based) with luciferase as reporter?

thanks,
Dvd]]> Tue, 24 Mar 2009 05:28:34 -0700 http://www.protocol-online.org/forums/index.php?showtopic=7104 http://www.protocol-online.org/forums/index.php?showtopic=6855 Thu, 12 Mar 2009 08:39:00 -0700 http://www.protocol-online.org/forums/index.php?showtopic=6855 http://www.protocol-online.org/forums/index.php?showtopic=6815 It has been always said that humans do not use more than 8% of the brain. If it is so, then like appendix, the unused portion of brain should have been vestigial and disappeared slowly during evolution. Can anybody explain to contradiction? Tue, 10 Mar 2009 02:49:02 -0700 http://www.protocol-online.org/forums/index.php?showtopic=6815