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here's the proto IH GFP Ab

DENISE at cellbio.emory.edu DENISE at cellbio.emory.edu
Thu Apr 8 15:37:18 EST 1999


GFPers,

Since Chris' e-mail on April 1st asking about GFP antibodies for immunohistochemistry, and 
my subsequent reply offering to send him the IH protocol I've used, I've received several 
e-mails requesting this protocol, so I'm posting here just in case it's helpful to others.

Cheers,
Denise Flaherty

Recall:  primary Ab being used-Chemicon's Chicken A'GFP (AB16901---$200 for 250ug).  
(Yes, Branwen, it's polyclonal-but my guess is that it will work nicely on your mouse slices--at 
least it's quite clean on the rat brain cells I've used it on).

IH FOR CELLS IN CULTURE  
(folks working with slices, you'll have to make your own appropriate modifications)

1. FIX
	2 X PBS pH7.4
	20 min / 4% Formaldehyde in PBS/ RT 	
		(*note: ~40% formaldehyde soln. diluted in PBS to 4%)
	3 X PBS pH7.4


2.  PERMEABLIZE/BLOCK
	20 min / 0.2% Triton X-100, 2% serum (whatever 2o is raised in), PBS / RT
		(*note: serum use 3-4 drops from Vector in 10 ml PBS)


3.  PRIMARY ANTIBODY	
	Overnight cold room in 2% serum (whatever 2o is raised in) , PBS
(for Chemicon's A'GFP--use 1:2500 [go 1:1500 if you think that your GFP signal is really low] 
in PBS with 2% Rabbit serum)

4.  WASH		
	3 X 10 min / PBS / RT


5.  SECONDARY ANTIBODY
	30min/ Vector secondary Ab / 2% serum (whatever 2o is raised in) PBS
		(for Mouse 1o MoAbs use HA'M-biotin 2o pre-absorbed with rat serum;
		for Rabbit 1o PoAbs use GA'R-biotin 2o pre-absorbed with rat serum)
(for Chemicon's chicken A'GFP, we use Rabbit A' Chicken-HRP [Sigma A-9792] 1:2500 in 
PBS with 2% Rabbit serum.  THEN GO RIGHT TO STEP 8.)

6.  WASH		
	3 X 10 min / PBS / RT
		(*note:  as soon as first wash is started,  prepare complex, it needs to sit 			30 min.  For 
Vectastain Elite Kit, use 2 drops of A, then 2 drops of B for every 5mls of PBS)


7.  COMPLEX (Avidin -Horseradish Peroxidase)
	30 min / RT


8.  WASH		
	3 X 10 min / PBS / RT (last one on ice)


9.  DAB		
	8 min / 1 tablet + 20 ml PBS + 5 ul H2O2 / ON_ICE 			
		(***DAB is a carcinogen make sure it is neutralized with bleach when 			done and that you 
wear gloves during use.  Bring tablet to RT before 			mixing and filter before use; add H2O2 
just prior to use)


10.  WASH		
	3 X 10 min / PBS / RT.  Store in PBS temporarily.




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