Double immunocytochemistry EXPRESS

[Jorge]

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Dear expert microscopists
I have the following query which I hope can help me.
I wish to make a double immunocytochemistry as quickly as possible,
preferring always to reduce over time the increase in noise or loss in
intensity.
Any advice of things that are possible to reduce or steps that I should
never reduce, will be very useful for me.
for example, increasing temperature, primary and secondary preincubate
increase the protein concentration in the lock solution, etc..
Obviously any suggestions I will have to standardize it to find the optimum
condition, but any ideas will be greatly appreciated
best regards

--
Jorge Toledo H.
Ph.D. student
Biomedical Neuroscience Institute (BNI)
Laboratory of Scientific Image Analysis (SCIAN-Lab)
Faculty of Medicine
University of Chile
www.scian.cl | www.couvelab.org

[Tim Feinstein-2]

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Hi Jorge,

If speed is your goal, start with a primary antibody directly conjugated with your fluorescent label (cy5, alexa488 etc).  This will cost more and take time to prepare in advance, but it cuts out half of the labeling process.  It has the added benefit of letting you use two different primary antibodies raised in the same species, but it limits your flexibility in label colors.  Incubating at high concentrations will let you get away with 10-15 min. of antibody labeling, naturally at the cost of nom-specific background.  In this case you could shave it to 15-30 min. Fix, depending on how you go about it, wash, 20 min. block (perhaps shorter if you use more serum/BSA?  I have not tried that), 15 min antibody in blocking medium and DAPI/Hoechst, wash, mount.  If going this way I would advise that you keep your washing steps pretty rigorous.  

Of course genetically encoded fluorophores need no staining at all.  I imagine that you already ruled that out.  

All the best,


TF

Timothy Feinstein, Ph.D.

On Aug 22, 2013, at 1:26 AM, Jorge <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear expert microscopists
> I have the following query which I hope can help me.
> I wish to make a double immunocytochemistry as quickly as possible,
> preferring always to reduce over time the increase in noise or loss in
> intensity.
> Any advice of things that are possible to reduce or steps that I should
> never reduce, will be very useful for me.
> for example, increasing temperature, primary and secondary preincubate
> increase the protein concentration in the lock solution, etc..
> Obviously any suggestions I will have to standardize it to find the optimum
> condition, but any ideas will be greatly appreciated
> best regards
>
> --
> Jorge Toledo H.
> Ph.D. student
> Biomedical Neuroscience Institute (BNI)
> Laboratory of Scientific Image Analysis (SCIAN-Lab)
> Faculty of Medicine
> University of Chile
> www.scian.cl | www.couvelab.org

[Jim Mansfield]

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Semi-commercial response:

Hi Jorge,

I am not an IHC expert (in fact I may be the only instructor at the National
Society of Histochemistry who does not stain slides!), but a colleague of
mine, Chris van der Loos, one of the leading experts on multicolor IHC,
wrote a guide to multiplexed immunohistochemistry that contains a lot of
information about double (and triple, and even quadruple) IHC methods that
you might find helpful:

http://www.biotechniques.com/multimedia/archive/00074/CRI-FP-Microscopy_7454
5a.pdf

There are also kits (and autostainer systems) available from Biocare, GBI
Labs, PerkinElmer, Ventana, Dako, Leica and more for doing dual-color
staining.

Good luck!

Jim


--

Jim Mansfield
[hidden email]
www.jmansfield.com
[phone]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Jorge
Sent: Thursday, August 22, 2013 1:26 AM
To: [hidden email]
Subject: Double immunocytochemistry EXPRESS

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Dear expert microscopists
I have the following query which I hope can help me.
I wish to make a double immunocytochemistry as quickly as possible,
preferring always to reduce over time the increase in noise or loss in
intensity.
Any advice of things that are possible to reduce or steps that I should
never reduce, will be very useful for me.
for example, increasing temperature, primary and secondary preincubate
increase the protein concentration in the lock solution, etc..
Obviously any suggestions I will have to standardize it to find the optimum
condition, but any ideas will be greatly appreciated best regards

--
Jorge Toledo H.
Ph.D. student
Biomedical Neuroscience Institute (BNI)
Laboratory of Scientific Image Analysis (SCIAN-Lab) Faculty of Medicine
University of Chile www.scian.cl | www.couvelab.org

[Martin Wessendorf-2]

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Dear Jorge--

On 8/22/2013 12:26 AM, Jorge wrote:

> I have the following query which I hope can help me.
> I wish to make a double immunocytochemistry as quickly as possible,
> preferring always to reduce over time the increase in noise or loss in
> intensity.
> Any advice of things that are possible to reduce or steps that I should
> never reduce, will be very useful for me.
> for example, increasing temperature, primary and secondary preincubate
> increase the protein concentration in the lock solution, etc..
> Obviously any suggestions I will have to standardize it to find the optimum
> condition, but any ideas will be greatly appreciated
> best regards

You might check the following reference:

Advanced laboratory techniques for sample processing and immunolabeling
using microwave radiation.
Ferris AM. Giberson RT. Sanders MA. Day JR.
Journal of Neuroscience Methods. 182(2):157-64, 2009

--Use of microwave radiation can apparently greatly speed up
immunocytochemical protocols.

Good luck!

Martin Wessendorf
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]