[Histonet] Re: Histonet Digest, Vol 112, Issue 22

[Mesru T]

Dear Histonetters,

I am going to do Immunofluoresence staining (beta-catenin, Notch1 and
Rnx2) on MMA sections of human iliac crest undecalcified tissue (5um thick).
In the literature I have encountered different protocols for removing the
MMA.
Neil Hand uses warm xylene, Nancy Troiano uses acetone and some other
people use the monomer itself. So many options to choose from and to get
confused. Does the deplasticizing protocol depends on the MMA embedding
method or section thickness? Is there are preferred method of
deplasticizing for IHC/IF staining?
Another confusion is the antigen retrieval. In some articles antigen
retrieval at boiling temperatures has been used, same as in paraffin
section IHC. Neil Hand recommends pressure cooker. However, Nancy Troiano
uses 50C water bath citric buffer (milder retrieval). Many researchers do
not even use antigen retrieval at all. I do not know which is the best
method to start optimization.
How does the type of antigen retrieval used for IHC depends on the MMA
method/polymerization time and temperature used?

*Brief MMA protocol:*


Activated Methyl Methacrylate (Monomer )

Methyl Methacrylate …………………… 200 ml

N-Butyl  Pthalate ………………………….. 50 ml

Benzoyl  Peroxide ……………………. .. 8.4 g

Store at 4C.



*Processing:*

**

70% ethanol ……………………………………….5 days

Absolute ethanol  ………………………………..  2-5 days

Xylene  …………………………………….... 2-5 days

Activated Methyl Methacrylate (Monomer )

                 ……………………………………...  6 days at 4C

Polymerize in 34ºC oven ……………………..  more then 6 days until hard.



Regards,

Mes