发信人: Marble (小石头哥哥), 信区: Biology
标 题: Re: 关于Immunohistochemistry的问题请教！
发信站: The unknown SPACE (Thu Jul 3 11:27:41 2003), 站内信件

agree. a good primary is essential to get decent IHC or IF.
typically for dilution of commercial product, i will start
from 1:50-1:500. do not try double labeling with another
antibody unless you are certain that the fluorchrome has
very distince exitation spectrum. frozen sections seem to
be used by Santa Crap all the times for IHC, while they would
tell you that it works for paraformaldehyde-fixed sections
if it indeed works. be aware that the purity of the fixative
(e.g., paraformaldehyde) is very critical to good signal
and less bgnd. do not overfix the tissue, and try antigen
retrieval with citric buffer all the time. incubation time
for 1Ab should be around 6-8hr, yet i have tried 2hrs for
abundantly expressed gene. for 2Ab, only 1hr should be enough.


【 在 Ras (很颓的猪。) 的大作中提到: 】
: 1: different concentration of Ab, say from 1:20-1000
: 2. differnt fluo-conjugated 2nd ab.
: 3. different fixation reagent. if using formaldehyde, try different
: pH (go as higha s 12)
: 4. different permeabilzing reagent, such as triton, saponin....
: after all, a good primary antibody is the key particularly if you
: are working on endogenouse proteins.
: 【 在 Oncogene (癌基因) 的大作中提到: 】
: : Then can you tell me what I should go to try first for
: : "different conditions"?
: : The time of 1st Ab, 2nd Ab, and the concentration of Abs?
: : How about the blocking solution? What do you usually use
: : for blocking?
: : Thanks alot.


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