[Histonet] Working with same host species primaries for double staining

[gayle callis]

We do sequential immunostaining to avoid cross reaction of a secondary
antibody to the next primary antibody, and also perform a normal serum block
that binds to the first secondary antibody: 

 

Two examples using two rat antimouse primaries and with immunofluorescent
method. This is done on fresh tissue frozen sections, so no NBF or PFA is
involved, eliminating the need for antigen retrieval. 

First normal serum block is matched to host of first secondary primary e.g.
donkey serum

First primary is rat antimouse

First secondary is donkey anti rat (F(ab')2 frag of IgG-fluorophore
conjugate

 SPECIAL NORMAL RAT SERUM BLOCK, 10% FOR 30 MINUTES.  This will bind to the
secondary antibody just used and eliminate a possible binding of this
secondary to the next primary antibody. Rinse very well. 

Second primary is rat antiMouse

#2 secondary antibody is can be a goat antiRat- fluorophore conjugate

Rinse very well.  

 

Our favorite method that works best for us, totally eliminating a secondary
antibody in the last sequence is:  

 

Normal serum block matched to secondary antibody

Streptavidin biotin kit block

First primary is rat antimouse

First secondary is donkey anti rat (F(ab')2 frag of IgG-fluorophore
conjugate

10% Normal rat serum block for 30 minutes as done above

Second primary antibody-biotinylated

Streptavidin-Alexa fluor dye

 

The best double staining method for enzyme immunohistochemistry where the
tissue is FFPE and needs two antigen retrieval is to use Chris van der Loos
method.  He published this in J of Histotechnology, September, 2008 and has
also presented a workshop using this method.  He is an expert on performing
double and even triple immunostaining and will be happy to discuss your
protocol with you and also send you a copy of his publication.
c.m.vanderloos <@t> amc.uva.nl 

 

Good luck 

 

Gayle Callis

HTL(ASCP)HT,MT

Bozeman MT