Hi arvind, this is for gary.
2009-02-12 TF 发件人: arvind 发送时间: 2009-02-12 14:29:58 收件人: tifei 抄送: 主题: Re: [Histonet] Antibody penetration problem try increasing the incubation time ant add 0.2 % triX to the primary i had also used the SMI 311 for my human brain sections it had worked well my sections were 50 micron and cryosections on slide On Thu, Feb 12, 2009 at 8:15 AM, TF <ti...@foxmail.com> wrote: just add up to 2% triton in antibody dilution and increase the incubation time. another way is to use floating sections. 2009-02-12 TF 发件人: Amos Brooks 发送时间: 2009-02-12 08:11:23 收件人: grudow1; histonet@lists.utsouthwestern.edu 抄送: 主题: [Histonet] Antibody penetration problem Hi Gary, I am certainly not an expert on doing this, but I have done it and it worked fairly well. Why not do this as a free floating section. Take the section and place it in a glass beaker to with xylene. Deparaffinize and rehydrate as you would a slide, but not on a slide ... in a beaker. Remember to agitate the section gently. Once you are out of xylene you might want to switch to a tissue culture flask (one of those flat rectangular ones with a cap). For antigen retrieval (always avoid the microwave) put the container in a 60 deg oven overnight (cap on of course). That should do the trick. You can always tweak the procedure later to improve results. Once you get to the antibody and detection you can use a capped tube on it's side if you keep it on a gentle shaker table. Once you have the tissue out of chromagen put it in a dish and float it onto a slide. Incidentally weather you do this all on a slide or in a jar to mount later you need to make sure there is sufficient surfactant in the buffer rinses. This really aids the penetration by removing the surface tension. Also make sure your pH is correct thru the whole procedure. Failing this will affect the antibody antigen affinity (or is it avidity ... I forget). Best of luck, (and let us know how it goes) Amos Brooks Message: 12 Date: Wed, 11 Feb 2009 15:19:09 -0500 From: Gay Rudow <grud...@jhmi.edu> Subject: [Histonet] Antibody penetration problem To: "'histo...@lists.utsouthwestern..edu'" <histonet@lists.utsouthwestern.edu> Message-ID: <864a73846ce8f04d995603351682c29c86a851c...@jhemtexvs2.win.ad.jhu.edu > Content-Type: text/plain; charset="iso-8859-7" I hate to change the topic, but I have a problem with penetration of an antibody. I am using 50 ěM paraffin sections and doing an antigen retrieval step of microwaving the sections in water for 5 min. I am staining by hand using the Vector ABC Elite mouse kit. The antibody is SMI 311 which I am using as a neuronal marker. Right now, my antibody penetration is only 15 ěM. Does anyone have any suggestions to help me with this? Thanks! Gay Rudow _______________________________________________ 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
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