[Histonet] p2y12 cryo rat problem high background

[Patpxs]

It could be the concentration of the antibody is too high.  Have you tried a lower dilution?

What species made the antibody?  I know that sounds a bit basic but I know that I have used a mouse or rat antibody by mistake.  

Paula

Sent from my iPhone

> On Nov 15, 2018, at 4:44 AM, Kooijman, E.J.M. (Esther) via Histonet <histonet at lists.utsouthwestern.edu> wrote:
> 
> Hello Bobbie,
> 
> But should I elimination endogenous peroxidase activity in brain/spinal cord tissues?
> Brains and spinal cord was harvested after cervical dislocation, then the tissue was snap frozen (isopentane..).
> 
> thanks for your help,
> Esther
> 
> -----Oorspronkelijk bericht-----
> Van: Boyce, Bobbie [mailto:Bobbie.Boyce at nemours.org] 
> Verzonden: donderdag 15 november 2018 12:21
> Aan: Kooijman, E.J.M. (Esther)
> Onderwerp: RE: p2y12 cryo rat problem high background
> 
> Hi Ester,
> Try Peroxoblock (Zymed) before your BSA block, but you have to be careful not to leave it on too long or it will eat your tissue. It's been a while since I've had to used it.
> 
> 
> Bobbie Boyce
> Histology Specialist III
> DuPont Experimental Station
> Nemours- Biomedical Research Department
> Histochemistry and Tissue Processing Core
> 200 Powder Mill Road, Bldg.400  Rm.5240
> Wilmington, DE 19803
> 
> (lab) 302-651-6771
> (fax) 302-651-5010
> 
> 
> 
> -----Original Message-----
> From: Kooijman, E.J.M. (Esther) via Histonet <histonet at lists.utsouthwestern.edu> 
> Sent: Thursday, November 15, 2018 5:53 AM
> To: histonet at lists.utsouthwestern.edu
> Subject: [Histonet] p2y12 cryo rat problem high background
> 
> **This is an External Email -  Please DO NOT open attachments or click links from unknown senders or unexpected email. **
> 
> Hello all,
> 
> 
> 
> I am trying to stain cryo brain sections from the rat 7um but having a lot of background. What am I doing wrong. Below the protocol is used and tried to adjust…
> 
> 
> 
> 1-      Fix the tissue with cold acetone (-20 ⁰C) for 10 minutes
> 
> 
> 
> 2-      Let the slide dry for 30 minutes at room temperature. Isolate the sections with DAKO pen
> 
> 
> 
> 3-      Block sections with BSA 2% in PBS for 1 hour at room temperature
> 
> 
> 
> 4-      Add primary antibody in PBS/BSA 1%, overnight at 4⁰C or 1h at room temperature
> 
> 
> 
> 5-      Wash 3x5 minutes with PBS/tween-20 0.05%
> 
> 
> 
> 6-      Add 100 µL Envision solution (goat anti-mouse/rabbit) and incubate for 1hr at RT
> 
> 
> 
> 7-      Wash 3x 5 minutes with PBS/tween-20 0.05%
> 
> 
> 
> 8-      Incubate with DAB (1:50) for 10 min (between 5-10 min; check color development)
> 
> (wear gloves, carcinogenic!)
> 
> 
> 
> 9-      Rinse thoroughly with miliQ water
> 
> 
> 
> 10-   Stain with haematoxylin for 1 min
> 
> 
> 
> 11-   Wash with running tap water for 5 min
> 
> 
> 
> 12-   Start the alcohol/xylene series (70% EtOH -> 80% EtOH -> 96% EtOH -> 100% EtOH -> 100% EtOH/xylene -> xylene -> xylene)
> 
> 
> 
> 13-   Mount slides with entallan
> 
> Kind regards,
> 
> 
> 
> 
> 
> Esther Kooijman  |  Research Technician  |  Department of Radiology and Nuclear medicine
> 
> The Netherlands
> 
> 
> 
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