[Histonet] Re: Histonet Digest, Vol 16, Issue 34

[Julia Edgar]

Prior to de-waxing, prepare a solution of 0.1% trypsin (e.g. Sigma-T-4799)
in 0.1% Calcium chloride and warm to 37oC.  It is important to make this
solution fresh, on the day of use.  Incubate sections for 20 minutes (or
whatever works for your particular antibody/antigen). Wash thoroughly in PBS
then proceed with blocking.

Julia

> I am doing immunofluorescence in paraffin sections. I have some
difficulties in it:
> 1: For pretreatment of tissue sections, I plan to incubate sections with
trypsin solution, but I do not know :what kind of concentration should I
choose ? (0.01% trypsin, 0.05% or o.1%)how long does it should last?
(10min,20min,30 min)
> 2: Recently, I read a protocol about double immunofluorescence, it shows
:Antibodies derived from different animal can be mixed and incubated as a
cocktail (example: rabbit anti-A and mouse anti-B). The same is valid for
secondary antibodies (example: goat anti-rabbit Texas Red conjugated and
goat anti-mouse fluorescein conjugated). but If secondary antibodies
cross-react, what should I do? My friend told me that I could use depletion
method. but I am not sure.
>
> Can anybody do me a favor?
> Thank you!
>
> Guofeng