Double label with same host antibodies

(for example, two rabbit antibodies)

 

            Sequential preparation

            First primary is run in serial dilutions and detected via TSA rxn (I use red)

            Second primary is detected via Alexa secondary (Alexa 488-green)

é      Of absolute necessity is to run controls:  most importantly, run slides in which

the second primary antibody is omitted but all other steps are included. This will determine whether there are any remaining open rabbit binding sites from the first primary to which the Alexa secondary is binding.  A clean control will show red label and no green label.  Also run slides in which the first primary is omitted but all other steps are included.  This will determine the level of non-specific binding of the detection steps of the first antibody.

                       

DAY 1             tissue is blocked with 3% H2O2 in 0.1M PB for 10 minutes

2x10 min PBS wash

1 hour block with 1% normal serum (I often use horse)

1° antibody incubation ON (over night) at 4°C

 

 

DAY 2             3x10 min PBS wash

 

Fab Biot/rb  1:1,000, 2hr  room temp

(an Fab secondary instead of an IgG secondary prevents an anti-rabbit arm from binding to the second primary-see diagram)

 

3x10 min PBS wash

ABC 2hr, room temp (make up ABC in PBS with 0.1% triton ~45 min prior to use)

3x10 min PBS wash

TSA rxn, time varies according to ab concentration

3x20 min PBST wash (minimum, I often wash ON to reduce background)

 

Fab/rb unlabelled 1:50, 2-4 hr room temp or ON 4°C

(When blocked with unlabelled FAB fragments after the TSA rxn, the controls without the second 1º are cleaner-helps to prevent any cross reactivity-see diagram)

 

 

DAY 3             3x10 min PBS wash

1 hr block with 1% normal serum

second 1º, normal working concentration, ON 4ºC

 

 

DAY 4             3x10 min PBS

Alexa 488/rb , 1:400, 2hr room temp

3x10 min PBS wash

coverslip with fluormount

 

The following antibodies I have used at various concentrations using the TSA red.  The optimal concentrations and TSA reaction times are as follows:

 

 

Antibody                Immuno dilution              TSA dilution and rxn time

Gustducin/rb                            1:1,000                                    1:20,000          13 min

  (Santa Cruz)

 

PLCβ2/rb                                1:1,000                                    1:7,000            10 min

  (Santa Cruz)

 

NGF/rb                                    1:500                                       1:2,000            7 min

  (Santa Cruz)                                                                                                          high background

                                                                                                1:5,000            10 min

                                                                                                                                 faint label

           

I would next try NGF/rb at 1:3,000 TSA rxn 8 min, 1:4,000 TSA rxn 10 min, and

            1:5,000 TSA rxn 13 min

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                                                        Bartel, September 2004