Can I use insulin primary antibody from cell signal technology, along with MACH 1 universal polymer detection system from biocare for immunohistochemical staining of rat paraffin embedded pancreaticislets of beta cells. I'm a medical.pathologist on research gate, please help.
Hi Madhan,
It depends on what animal your insulin primary Ab was generated in. The product information sheet for the Mach 1 universal (link below) indicates it will react with Mouse IgG, Mouse IgM and Rabbit IgG heavy and light chains. That would encompass most mouse and rabbit primaries; but the specifics of your primary should be listed on its product sheet.
http://biocare.net/wp-content/uploads/M1U539.pdf
if you don't know how to check and you provide details for your primary - a product number or something - I'd be happy to look into it for you.
Chris
thanks dr.christopher, primary Ab generated from rabbit only,the product code is 4590S, from cell signal technology,which has species reactivity for rat tissue, as i am going to use it on rat paraffin sections.
madhan
no problem. The Mach 1 won't recognize a rabbit primary. Cell signaling has a anit-rabbit HRP Ab but it isn't certified for IHC. Here's a link http://www.cellsignal.com/products/secondary-antibodies/7074?Ntt=rabbit+hrp&fromPage=plp
I would suggest trying a different one. This one from Life technologies should do the trick - http://www.lifetechnologies.com/order/catalog/product/656120
It is an HRP conjugate so it should work with the same protocol you're using for the cell signaling Abs you would just have to dilute the secondary AB in the same buffer as you use for the primary because it isn't pre-diluted like the MACH 1. Everything else should be the same. I generally start with a 1:1000 dilution for secondaries and adjust if necessary, 90% of the time 1:1000 works well.
Hope that helps.
Chris
once again thanks chris,the product code 4590 S, i gave has application for IHC -P also.thats what the protocol from cell signal says.
Hi Madhan, your primary will work just fine for IHC, most likely with with the cell signaling secondary antibody I gave a link to above. But that secondary (the cell signaling anti rabbit HRP conjugate) has not been certified for use in IHC, only on western blots. So I suggest trying a different one, the life technologies secondary, because this one has been tested by the manufacture for IHC on FFPE tissue.
Again, if it were me, I would use your primary with this secondary - http://www.lifetechnologies.com/order/catalog/product/656120 and I would dilute the secondary 1:1000 before use.
Hi chris,my question is whether primary antibody from cell signal (4590 S) will work with MACH 1 or not?else should i go with lifetechnologies secondary Ab, as suggested by you? please also tell me which primary insulin antibody will react well with MACH 1 on paraffin embedded pancreatic rat tissue sections?as i have MACH 1 only in our pathology lab,as u know my project is a self funded one.please help me out to find one.thanks.
madhan
Test it first. I work primary with mouse and rat tissues and generally use rabbit primary antibodies for both species.
It is very difficult to say which primary will react with MACH1. There is very sparse information of this secondary system on the web. Can you upload a datasheet of the MACH1 system. If it is a universal anti-Rabbit-Mouse-Rat , then it is very likely to get background on rat samples.
Cheers,
Ivan