[Histonet] p2y12 cryo rat problem high background

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

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