Help to provide immunoflourence protocol to paraffin embedded slides

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nalpy
nalpy's picture
Help to provide immunoflourence protocol to paraffin embedded slides

Dear friends

Pls I need indirect immunoflourscence protocol to paraffin embedded slides. i confuse whether do antigen retrival in immunoflourscence? because paper protocol not mention this.pls clarify this to me. I plan to take photograph to confocal microscope, i need mounting medium preparation for immunoflourscence.
Thank you

Arvind Singh Pundir
Arvind Singh Pundir's picture
Hi Nalpy,there are many

Hi Nalpy,
there are many protocols in protocol section of this website(Scientist solutions.com)
some of them are

Antigen Retrieval depends on your tissue state whether your primary antibody is accessible to antigens or not , because certain conditions(fixation , temp, and certain harsh conditions) block your Antigen epitope thereby preventing your primary antibody to bind to that , there you have to do antigen retrieval to expose those epitopes, in parrafin sections i will recomend you to do antigen retrieval, there are many methods available , try the one which best suites your tissue and  protocol conditions, regarding mounting media you have to use aqueous antifading mounting media like Vectashield..

samm
samm's picture
See Pundir's post for antigen

See Pundir's post for antigen retreival. Note that there are many methods (pressure cooking, microwaving, enzyme treatment) and buffers for Ag retreival - choosing among them is often a trial-and error process.
As for the mounting medium, you need one that contains anti-fade agents, especially if you want to do any confocal or spinning disk IF for acceptible acquisition periods. Invitrogen's Prolong Gold, Calbiochem's FluorSav and Vector Labs Vectashield 1000 series are good broad range bets. Others, such as the one by Sigma work well for some conjugates, but not others. You can also make a homemade prep for basic use, though I'd strongly recommend one of the former bunch if you plan to do confocal.