Hi! I have purchased Nrf2 Abcam antibody (ab31163). I want to perform an inmunohistochemistry in paraformaldehyde fixed rat brain sections. I have not clear which protocol should I follow, inmunofluorescence with Alexa 488 as secondary antibody or another IHC protocol with DAB visualization. Is it neccesary antigen retrieval for any of this protocols?
Thank you in advanced ;)
Sara
Hi, Sara.
IHC and IF both are useful tools for morphological studies, but there are several differences. The decision on which protocol should be used should depend on the purpose and requirement of experiment.
Here are IF protocol used in my lab:
1. Free floating section: http://www.creative-diagnostics.com/immunofluorescence-protocol-free-floating-section.htm
2. stick section: http://www.creative-diagnostics.com/immunofluorescence-protocol-stick-section.htm
As fixation in a solution of paraformaldehyd causes some unspecific background fluorescences I would recommend DAB visualization including controls.
Regards, Fred
Hi all, thank you for your answers and advices I took them all and I did a probe with inmunofluoresce and with the DAB reaction. After seeing both images in the microscope (images enclosed, 20x) I discared the inmunofluorescence and I went on with DAB. Analyzing the images in the microscope I have realized thet there are "two different" types of cells, I am not sure if they are two different types of cells or just the ones with blue cicle are precipitates of the DAB reation. I am pretty sure that the ones with the red circle are the cells positive for NRf2 receptor but I do not know 100% if I am ok or wrong. Would you possible confirm me this? What do you think about this "two differente type of cells"? Thak you in adanced ;)
Best,
Sara.
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