[Histonet] p2y12 cryo rat problem high background

[Kooijman, E.J.M. (Esther)]

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

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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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