[Histonet] Uneven fluorescent staining

Tue Jun 15 10:28:42 CDT 2010

Thank you so much for your answer Anatoli!
- I  tried the citrate buffer boiling system and I obtained the same unreliable results with NeuN. Some animals showed a great NeuN staining and others did not. I really thought at that point that my perfusion was the problem and I switched from making my own PFA4% to buying a 16%PFA from Electron Microscopy Sciences, which I think solved the problem. Now I'm obtaining a good NeuN staining but only in some regions of the tissue. 
- I am using the Rat monoclonal from Abcam (1:250)
- I'll consider the new method using Edu labeling for new experiment and will try the ProLong Gold antifade, thank you very much for the advice.
 

Alexia Nunez
University of Maryland, College Park


> Subject: RE: [Histonet] Uneven fluorescent staining
> Date: Tue, 15 Jun 2010 10:26:32 -0400
> From: AGleiberman <@t> cbiolabs.com
> To: alexia_fran <@t> hotmail.com
> 
> Alexia,
> 1. HCl treatment could be sometimes problematic - many epitopes are quite sensitive to it, so your NeuN antibody will work not in optimal conditions. You can replace acid treatment with citrate buffer boiling - the same treatment that is used for paraffin sections. It will denature DNA as effectively as HCl treatment.
> 2. You did not mention what antibody you are using for BrdU - the best are rat monoclonal from Abcam (much better than any mouse monoclonal or sheep or goat polyclonal I have tested) - and you can easy combine them with mouse monoclonal against NeuN (I believe, only mouse anti-NeuN exist).
> 3. For future experiments I recommend switch from BrdU labeling to EdU labeling (Click-iT® EdU Alexa Fluor® 488 HCS Assay from Invitrogen) - this technique is more sensitive for studying DNA synthesis than BrdU, staining is faster (30min), does not involve neither denaturation step nor antibody and can be easily combined with any other marker staining. To do antigen staining after EdU visualization, that is simple chemical reaction, you need wash thoroughly your slides and perform your antibody staining. Additional benefit is - much better structure of nuclei because denaturation step sometimes affects them. In addition, because of high sensitivity you can decrease concentration of nucleotide substitute (EdU) 5-10 times in comparison with BrdU that can be crucial for long-term experiments because all these nucleotide analogues (especially BrdU) can affect cell fate in long run.
> 4. Finally, I recommend to replace Vectashield with ProLong Gold antifade (Invitrogen) with or without DAPI. It is better for fluorochrome preservation and it will harden overnight - you don't need any nail polish
> 
> 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 Alexia Francisca Núñez Parra
> Sent: Monday, June 14, 2010 6:10 PM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Uneven fluorescent staining
> 
> 
> Hello everyone!! 
> The Histonet Archives have been always very helpful to me, so I decided to become a member and ask a personal question to the experts.
> 
> I am doing NeuN and BrdU double staining in the olfactory bulb, following the protocol described below. I have always had some trouble with the NeuN staining, obtaining sometimes good or no staining at all. I tried to trouble shoot with the intracardial perfusion and even bought a new NeuN antibody (Chemicon). I am having now, however, another problem. I observed an uneven NeuN and BrdU staining. There are some parts of the section that get a really good and bright staining and others that do not get any staining at all (even though they should).
> 
> Can anyone give a hint on what to review or change?
> 
> Thank you so much!!!
> 
> Alexia 
> 
> 1. Intracardial perfusion with cold PBS and cold 4%PFA.
> 2. The brains are postfixed for 4 hrs in PFA 4% at 4C and immersed in sucrose 30% ON. After that, the brains are embedded in tissue tek and store at -80.
> 3. Sections of 20um are obtained using a cryostat and collected using positive charged slides.
> 
> 
> 
> 
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> 
> 
> 
> 
> 
> 
> 
> 
> 4.    
> Allow the slides to reach the RT 
> 
> 
> 5.    
> Warm slides at 55C x 10'
> 
> 6.    
> Outline slides with Immedge pen
> 
> 7.    
> Hydrate: PBS 2 x 5', final quick wash with ddH20
> 
> 8.    
> Soak slides in 2N HCl for 1 hr at 37C
> 
> 9.    
> Neutralize washing the slides with 0.1M Na tretaborate buffer
> pH8.5, 3 x 10'
> 
> 10.    
> Wash with PBS 2 x 10'
> 
> 11. 
> Wash with PBS-T 1 x 10'
> 
> 
> 
> 12.    
> Block at RT with 10% of Donkey serum in PBST x 2hrs (60uL on each section)
> 
> 13.    
> First antibody: incubate at 4C with first antibody diluited in
> PBST with 2.5% Donkey serum ON (16-24hrs)
> 
> 14.    
> Wash with PBST 4x10' at RT 
> 
> 15.    
> Secondary antibody: dilute 1:750 alexa antibodies in PBST with
> 2.5% Donkey serum x 2 hrs at RT
> 
> 16.    
> Wash with PBST 1 x 10', and then with PBS 3x 10'
> 
> 
> 
> 17.    
> Mount with Vectashield and seal using transparent nail polish. 
> 
> 
> 
> 
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