[Histonet] Uneven fluorescent staining

[Alexia Francisca Núñez Parra]

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.














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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