Staining of E15.5 mouse embryo brain tissue

[JDenbeig]

Hello,

I am having difficulty with my staining of E15.5 mouse embryos and would appreciate some help.
I am trying to stain for VEGFR2 and Endoglin (separately) in embryo brain tissue using a Perkin Elmer tyramide amplification kit.

At the moment, I see a significant amount of staining for both the Rat Isotype Control antibody as well as anything involving the tyramide steps.  To combat this I increased the peroxidase quenching time from 15 mintues to 1 hr, but this hasn't produced a significant decrease in background.  I have also looked at the Tyramide Kit from Invitrogen, but so far no change.

On top of this, I am seeing huge amounts of staining in the liver.  My tissues are not perfused so perhaps this is from the blood cells? Regardless, the auto-fluorescence or non specific staining of this tissue is frustrating.

Because my proteins of interest have such low expression, any background is really compromising my stain, as the ultimate goal is to quantify the amount of VEGFR2 and Endoglin receptor in my tissue.  I know that VEGFR2 is difficult to identify - does anyone have suggestions on antibodies (including isotype control) or changes to my technique?  I have included my protocol below.  My next approach is to try a brain specific antibody (from posts on these boards I suspect GFAP may be a good choice) to see whether this works as a positive control.

Any advice would be much appreciated.

Thanks so much,
Janet

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My protocol is as follows:

Antibodies:
VEGFR2 Biotin-anti-mouse (monoclonal eBioscience @1:400)
Endoglin Biotin-anti-mouse (monoclonal eBioscience @1:400)
Rat Isotype Biotin-anti-mouse (monoclonal eBioscience @1:400)
Streptavidin HRP (Perkin Elmer @1:100)
Tyramide Cy3 amplification (Perkin Elmer @1:50)

Fixation:
- embryos are dissected out and fixed in 4% PFA for at least 24hrs
- after fixation, embryos are stored in 70%EtOH until paraffinized
- 5 um slices of tissue are prepared on slides (from paraffin)

Deparaffinization, Antigen retrieval and Quenching:
- 5 min xylenes (x2)
- 100% EtOH, 100%EtOH, 95%EtOH, 70%EtOH, H2O, PBS (x2)  (5 min each)
- 10 min Proteinase K [@ 20 ug/mL]
- 5 min PBT  (PBS + Tween) wash
- 3% H2O2 in PBS for 1 hr

Blocking and Antibodies:
- 1hr (x2) using Blocking Solution (BS): 20% Heat Inactivated Sheep Serum, 2% Donkey Serum, 1% Heat Inactivated Fetal Calf Serum
- Block endogenous avidin and biotin with Vector ABC kit (PK-6100) - 30 min each
- Wash in PBST (PBS with 1% Tris) 5 min (x3)
- 1st Ab reaction (VEGFR2, Endoglin or Rat Isotype) in 20% BS (as above) at 4C overnight
- Slides are placed in humidified box and covered with parafilm to keep moist.
- Wash in 2% Blocking solution for 1 hr (5x)
- 2nd Ab (Streptavidin HRP) @ 1:100 in 20% Block Solution overnight at 4C
- 2% Block Solution washes (2hr)
- TNT washes (30 min, rt)
- Tyramide Amplification (1hr incubation)
- TNT wash
- Stop reaction with 3% H2O2 in TNT
- Wash overnight in TNT, 4C
- Glycerol wash to make tissue transparent
- Dehydration in H20, 50%EtOH, 70%EtOH, 95% EtOH, 100%EtOH
- Slip cover with Dako fluorecence medium


I have conducted the following controls:

Rat Isotype control
Streptavidin-HRP only
Streptavidin-HRP + Tyramide
Tyramide only
No stain

At this point all tyramide stained slides produce some level of background, including the rat isotype control.

[CanuckPhD]

I agree that your background may be due to endogenous biotin. Are you using the the ABC kit to block or the Vector Avidin-biotin blocking kit (SP-2001)? To test this just try incubating your tissue with ABC after peroxidase quenching and serum block. If you get positive staining this suggest this is the cause. As well, to check if your biotin blocking is adequate add some fluorescent labelled avidin after your biotin block step. There should be very little staining. If the biotin block isn't good then you might want to go away from avidin biotin based methods and try something like Dako's Envison kit.

Your idea to perform a GFAP stain is good to determine if it is just these antibodies or your protocol. Overall I think your protocol looks fine. Have you tried using different types of antigen retrieval? I have found that Proteinase K causes more background then just citrate buffer pH 6.0 at 98 degrees.

[JDenbeig]

Terrific.

I will test the biotin blocking as well as the GFAP and post back.
I'll have to do a bit of research into the citrate buffer option. 
Thanks for the suggestions.