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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
