2-methyl butane - immunocytochemistry (Oct/23/2003 )

hy, i am new in immunocytochemistry and i am trying to evidentiate with a polyclonal (rabbit) primary antibody produced against a 20 aa peptide a nuclear protein present in the retina cells in mouse embryos.
all the info i have from a previous published paper (from others) that used a different antibody (not available) against this same protein is that they used 2-methyl butane to add the penetration of the antibody, then they incubated 10 days at 4°C with the primary antibody, then 2 days at 4°C with the secondary then again 2 days with streptavidin...
my questions are..do u ever used 2 methyl butane?
is it the same thing as using microwave to make antibody enter the nuclei?
is it 10 days incubation a little bit exaggerated (as 2 days with secondary antibody and 2 days with streptavidin) or you also ever used so long times?
how many changes do i have that an antibody raised (in a company) against a 20 aa peptide and checked with elisa with excellent results doesnt work in immunocytochemistry? (i tried it with over weekend incubations on embryo slides and no signal was evidentiated..)

i thank you for your kind reply

Daniela

Hi Daniela

Unfortunately ELISA against the peptide doesn't really mean it will see the native protein.
I had similar problems which is why I opted to have a recombinant protein expressed by HSL.
Personally I think the time scale you were talking about seems very long... the longest we use is 18 hours.

Unfortunately I am unable to comment on methyl butane.

If you need any advice about maybe looking at having a protein expressed please feel free to contact me on here or on dynamite_davis@hotmail.com

I'm kind of reluctant to talk about companies and collaborations on here as this is more about sharing experiences and technical advice rather than giving my suppliers a boost in sales!

I hope I have helped a little

Regards
Dynamite