[Histonet] p2y12 cryo rat problem high background
Kooijman, E.J.M. (Esther)
e.kooijman at vumc.nl
Thu Nov 15 04:53:04 CST 2018
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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