Maria,  thank you very much for the suggestions.

> I usually air-dry the sections @ RT as I'm cutting. After your
> 50 sections are cut, then freeze store @ -70C those sections not
> being used for IHC
> You should'nt have to re-fix the tissue after cutting,
> because you have already perfused (successfully) and fixed the
> tissue for additional time - this is enough!

You and others have pointed out that I do not have to refix the section
on the slide before the storage.  I will follow your instruction!

> In your IHC protocol have you a need for antigen retrieval. 

How often do you use antigen retrieval for the frozen sample?  I thought
the antigen retrieval was for the paraffin section.

> > for blocking can use 5% normal serum - 30mins or
>   for 1hr. or can use 10% normal serum - 30mins
> but, I have stopped using normal serum as a blocker and do not use
> normal serum in with antibodies or in washes, because sometimes
> the cause for background comes from the use of normal serum. So,
> I use non-serum blockers and there are many kinds on the market.
> I use one agent for background blocking from Innovex Biosciences
> product # NB306. It's ready to use for 8um-20um sections 10-15mins.
> For thicker sections 20mins w/ agitation. 

This is very interesting.  So for mice, non-seerum blocker gives
superior results than normal goat serum?  Do you wash with PBS in each
step?

> Since, I don't know what antigen your trying to label I can't help
> you w/ this step, except that I just make my antibody w/ DPBS -
> no serum added for 30mins - overnight @ RT w/ agitation.

What is DPBS?  Is it PBS?  Don't you put BSA in PBS for washing
solution?

> And the same for secondary antibody (in my opinion, some of the best
> secondary antibodies come from Jackson labs) 30mins - 1hrs @ RT
> (depends on how thick the tissue is and the standard dilutions are
> 1:200-1:400). 

Another basic question.  What kind of secondary antibody, or primary
anibody is the best for mice?  rabbit primary antibody and Fit-C
conjugated goat antirabbit secondary antibody are the those I use.   I
always wonder if I should chose polyclonal or monoclonal, rabbit or goat
primary antibody, goat or rabbit antibody?

Thank you very much, Maria,  for kind suggestion in advance.  Please
give me more suggestion if you are not tired.   I will treat you with
SUSHI when I visit San Fransisco.

-- 
Masayuki Miyagishima, MD
University of Pittsburgh, 
Department of Surgery
412-647-2345, and ask the hospital operator to page 3228, please.

Maria Mejia wrote:
> 
> Hello from San Francisco! I've never done IHC on heart nor vessel
> tissue, but I do a lot of IHC on brain and retina tissue. I use
> either frozen sections, free-floating or paraffin sections. So,
> lets see if I understand you correctly, when you perfuse you use
> freshly made 4% PFA (cold) and remove the tissue and further fix
> (again in cold) 4% PFA for an additional - 2hrs. Then cryoprotect
> in 30% sucrose @ 4C - overnight. Next day you freeze tissue and
> place in -70C freezer. When you cut tissue how thick are the
> sections? If you are cutting frozen sections (8-20um) and pick-
> up tissue on warm glass slides (that's gelatin alum-coated slides)
> I usually air-dry the sections @ RT as I'm cutting. After your
> 50 sections are cut, then freeze store @ -70C those sections not
> being used for IHC and those for IHC can do IHC on the same day
> as sectioning or store these slides in the refrig @ 4C 24-48hrs.
> But, these slides should be stored in a slide box with some
> wet (use H20) paper to create a humid condition and then seal the
> box w/ tape. You should'nt have to re-fix the tissue after cutting,
> because you have already perfused (successfully) and fixed the
> tissue for additional time - this is enough!
> If you use the sections stored in the -70 C freezer for IHC don't
> forget to let these sections come to room temp before IHC.
> In your IHC protocol have you a need for antigen retrieval. That
> is, are you having success w/ your IHC protocol w/out AR, if not,
> may I suggest you do antigen retrieval before blocking step -
> here's what I use (may or may not work on your antibodies).
> > antigen retrievel sol. 0.1M Tris buffer @ RT - 30mins-1hr
>   Tris buffer (Trizma HCl Sigma T3253) 0.8gm
>                                  DH20 63ml
>   Make fresh and store @ RT. Sol. stable for 24-36hrs @ RT.
> 
> > for blocking can use 5% normal serum - 30mins or
>   for 1hr. or can use 10% normal serum - 30mins
> but, I have stopped using normal serum as a blocker and do not use
> normal serum in with antibodies or in washes, because sometimes
> the cause for background comes from the use of normal serum. So,
> I use non-serum blockers and there are many kinds on the market.
> I use one agent for background blocking from Innovex Biosciences
> product # NB306. It's ready to use for 8um-20um sections 10-15mins.
> For thicker sections 20mins w/ agitation. Now, as you know this
> type of blocking is not for endogenous peroxidase material for that
> you will have to treat tissue w/ 3% H202 in methanol or water -
> 10-20mins before blocking tissue.
> Since, I don't know what antigen your trying to label I can't help
> you w/ this step, except that I just make my antibody w/ DPBS -
> no serum added for 30mins - overnight @ RT w/ agitation.
> And the same for secondary antibody (in my opinion, some of the best
> secondary antibodies come from Jackson labs) 30mins - 1hrs @ RT
> (depends on how thick the tissue is and the standard dilutions are
> 1:200-1:400). I hope this information helps you. Please let me
> know if I can help further.
> maria mejia
> smith-kettlewell eye res. inst.