Using Western Antibodies for IHC - General rules- suggestions (Oct/02/2006 )
Hello,
I am trying to standardise IHC for a protien of interest that I have already standardised western for.
I have the AB that I use for western and I am hoping to use the same for IHC too. As I understand I dont think I should have a problem about that. But I need a few clarifications before I embark, so here are the question
1. Are there any absolute contra-indications?
2. Are there any general rules about AB dilution or must this be standardised for each AB?
3. CYP1B1 is my protien of interest, and I am looking at it in FFPE rat tissues if anybody has worked with this before, do you recommend an antigen retrieval step and if so what is it? (also, I would be exteremely grateful if you can PM a protocol to me!)
4. WHat about secondary AB?
Thank you in advance for your suggestions.
I am trying to standardise IHC for a protien of interest that I have already standardised western for.
I have the AB that I use for western and I am hoping to use the same for IHC too. As I understand I dont think I should have a problem about that. But I need a few clarifications before I embark, so here are the question
1. Are there any absolute contra-indications?
2. Are there any general rules about AB dilution or must this be standardised for each AB?
3. CYP1B1 is my protien of interest, and I am looking at it in FFPE rat tissues if anybody has worked with this before, do you recommend an antigen retrieval step and if so what is it? (also, I would be exteremely grateful if you can PM a protocol to me!)
4. WHat about secondary AB?
Thank you in advance for your suggestions.
a lot of questions; we sometimes have similar questions to Ab; if for your Wb-used Ab there is no information if it it will work in IHC, ask your provider (if it is commercial; sometimes, manufacturer offer a deal: You test its Ab for a special, untested application, here: IHC, and you get the Ab for the half price if you tell the firm your results); if no-one has tested before, you have to try; it may work...
Ab concentrations for IHC are about 10- to 50-times more concentrated as for Wb (start with 1:100 for IHC if you use 1:1000 for WB)
as secondary Ab use Alexa-fluor-labeled Ab, they shine brighter than FITC, TRITC or Cy
Your Ab will work if it can recognize the epitope when the protein is in its native form
U need to use a higher conc. of antibody when compared to westerns.
I am not sure if u want to use antigen retrieval or not. But we always do it for all IHC.
TBS-antigen retrival sol -1L
2.42 gms of Tris Base
8 gms of NaCl
pH 9.0 ( antigen retrieval)
We leave it for 4-6 hrs at 60C with the antigen retrival sol. before blocking the sections.