Srinivas

sorry for the delay in getting back to you.

I will outline the technique listed in Fluorescent Protein Tracing by Nairn that I cited in last message to you.

before doing so, I am not convinced from the message you posted yesterday that the problem is in your primary antibody.

I recommend that you omit the primary antibody on a known positive control slide, substituting the buffer you use for rinsing your slides and incubate along with another slide that receives the primary antibody.

If you are getting positive staining on the slide that had buffer instead of the primary antibody, you can conclude that the secondary antibody (goat anti-rabbit) may be the problem

also, are you making certain that your sections never dry out once the method has begun? this will be essential to avoiding any non-specific staining

since you observe the problem with two different primary antibodies from two different sources (one home made and the other purchase commercially) I am inclined to think that your problem is not caused by the primaries so I recommend that you look elsewhere before embarking on the absorption. as you will see below, the method of preparing the sheep liver is quite involved and time consuming.

if you are using an avidin-biotin detection system it is critical that you perform a biotin blocking step to avoid non-specific staining.

the method for liver is as follows:
wash the organ or tissue clear of blood with physiologic saline, dice with scissors or knife and wash again with saline.
the diced material is then mixed with an equal volume of cold saline and homogenized without allowing excessive warming. the product is frozen at -76 degrees C and thawed to facilitate centrifuging and washing. centrifuge at 4000 g for 10 minutes and wash the cellular material twice with phosphate buffered saline

this suspension is centrifuged at 10,000g for 15 minutes in separate amounts designed to give 0.5 ml or 1.0 ml volumes of packed tissue in each centrifuge tube, which is drained, stoppered and frozen at -20 degrees C.

absoprtion is carried out by stirring each ml of antibody with 0.5 ml of thawed homogenate, incubating at 37 degrees C for 30-60 minutes and leaving to stand overnight at 0- -2 degrees C.

alternately, antibody and homogenate may be shaken for 2 hours at room temperature. the antibody is recovered by centrifuging at 10,000 g for 20 minutes

the powders are prepared either by lyophilization of homogenates or by acetone drying. for the latter, wash the diced liver and gind with acetone in a mortar and pestle or low speed homogenizer. the product is filtered through coarse filter paper. the deposit on the paper is washed several times with acetone to complete the dehydration and then dried at 37 degrees C or overnight at room temperature.
the dried tissue is ground to powder in a mortar or mechanical grinder and sieved through a 1 mm wire mesh to remove coarse debris. the dehydrated  powder may be stored at room temperature until required. 

as stated in my last message, for absoption of your antibody,  the ratio of liver powder to antibody does not need to be precise, but more is better than less to ensure complete absorption. the ratio of 100 mg of liver to 1 ml of antibody is given in the reference. if you have 0.5 ml of antibody you would use 50 mg of liver powder etc.

good luck


Vinnie Della Speranza
Manager for Anatomic Pathology Services
Medical University of South Carolina
165 Ashley Avenue  Suite 309
Charleston, SC 29425
Ph: 843-792-6353
fax: 843-792-8974

>>> Mr srinivas sv  05/03/05 12:20AM >>>

Thanks Ms. Vinnie Della Speranza and Mr. kharazia for the reply.

Can anyone tell me the protocol for preparing the liver acetone powder, which i am planning to use as an adsorbent to non specific agents in a polyclonal antibody. 

I am looking at the expression of steroidogenic enzymes in sheep ovary. I think most have heard of my problem in my previous mails. To tell briefly, I am getting same color reaction in both positive and negative controls. so i am planning to try the adsorbtion technique using liver acetone powder. There is no commercial preparation of  liver acetone powder from sheep. only liver powder from calf, rat are available. it would help me, if anyone tell me which one i can use or the protocol for preparation of sheep liver acetone powder as i have got sheep liver with me.

Also i need to know the concentration of the liver powder i should use with the antibodies.

Thanks

waiting for the replies..

Srinivas

 


Srinivas Seekallu 
PhD student
Dept of Vet Biomed.Sci.
Western College of Veterinary Medicine,
University of Saskatchewan.
Saskatoon, Canada.

Ph: 306-477-0849 (Home)
    306-966-7373 (Lab)
    306-966-7382 (Office)
		
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