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@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.bo...@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@lists.utsouthwestern.edu> > Sent: Thursday, November 15, 2018 5:53 AM > To: histonet@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 > > > > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.utsouthwestern.edu_mailman_listinfo_histonet&d=DwIGaQ&c=X2IGR6v8ax_mLhSmU1r3Aw&r=NAc-sZTcJtcdymsLif_fp7ngB_dNBpF-UI64u9fuosc&m=N05bQ_qJ2Li62f83kZl-FK7rp5Ad-spj9JTvwovnnKQ&s=6MYtZ-ICVPPxaohoUjidJjm_mNhwB7nDA1Np5SRxT4w&e= > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
