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


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.

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).

(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

	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)

	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

	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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