[Histonet] i need some technical help!

[Mr srinivas sv]

Hello Vinnie and all users of histonet,
 
Thanks for the help.I think i was not clear in my mail. I am looking at the expression of steroidogenic enzymes in sheep ovary at different physiological status. my primary antibodies are polyclonal rabbit anti-mouse and my seconadary is Goat anti rabbit conjugated with HRP. one primary antibody is home grown but from different lab and the other is commercial from Research diagnostics. My seconadary is not biotilynated and i am using VIP ( has H2O2 as a substrate) as a color reagent. I am using 1% H2O2 to block the endogenous peroxides and Normal goat serum with 1%BSA for blocking the nonspecific antigens (Serum blocking). I am using Normal Rabbit sera for negative control as my primary antibody is raised in rabbit. 
I have reached the dilution of primary antibody to work with by looking at  the response obtained with different dilution and 1:200 dilutions works good for the primary and 1:100 dilution works good for the secondary antibody. my problem is that i am getting the color reaction in both positive ( ACTUAL PRIMARY Ab) as well my negative (NORMAL RABBIT SERA) controls. so the problem is with my normal rabbit sera (source from SIGMA) . i beleive that there is less problem with the background staining. I am facing problem with both the home grown as well the commercial source of antibodies.
 
so my tissue sections are cryosections and are from sheep. but i am using the primary antibody raised in rabbit. i am planning to use the liver acetone powder with the idea of blocking non specific sites in my tissues or blocking non specific antibodies in my polyclonal primary antibody raised in rabbit and the normal rabbit sera(NRS) which i am using as a negative control. i am not sure about the source of the liver acetone powder i need to use. unfortunately sheep liver powder is not available commercially. so i was planning to go for the calf one. i have not yet ordered and waiting for your suggestions. but as you suggested, i have got the sheep liver with me and it would be of great help if you let me know how to prepare the sheep liver acetone powder by my own. whether it helps to correct my problem???
 
Also, for your information,  i am not left with 1ml of  primary antibody but have sufficient NRS.
 
or would you suggest me any alteration in my protocol??? 
 
Thanks in advance for any kind of help. This is my first trial of IHC and am planning to do lots in future. I enjoy doing it and learning from the mistakes and trouble shooting the problem. but it will be fun if antibodies, time and the cost is not the constraint.
 
Thanks once again. 
 
waiting for your reply
 
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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