Re: membrane/cytoplasmatic ratio *vendor response*

[Kilgore, Jason A.]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

** Vendor Response **

I will second Mel's response.

I've had good luck using wheat germ agglutinin (WGA) conjugates for plasma membrane (PM) labeling (I wrote the Molecular Probes manual for it when I was in R&D here, which has a protocol).  You can label prior to fixation, but beware that normal endocytosis will lead to some internalization.  Or you can label after formaldehyde fixation (I haven't seen any entry of the dye upon labeling after fixation, but be sure to use methanol-free formaldehyde stock -- no "formalin").  Don't permeabilize until after doing the fix and label, or the WGA will label internal structures.  These points are all true for Concanavalin A (ConA) conjugates, too.

As Mel pointed out, lipophilic cyanine dyes like DiI and CM-DiI C18 will label internal membranes as well as PM, and will be lost with permeabilization.  Do not use the FM dyes (as they are internalized much too quickly).

Another choice is to label with CellMask plasma membrane stains, but those won't survive permeabilization, if needed.

Jason
Molecular Probes


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Menelaos Symeonides
Sent: Monday, March 02, 2015 8:08 AM
To: [hidden email]
Subject: Re: membrane/cytoplasmatic ratio

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Valeria,

I think George covered line scan analysis pretty well. If I'm understanding your question correctly, you are also looking for a formaldehyde-fixable membrane stain so that you can localize your GFP moiety in comparison to it rather than just looking for the outer edge of the cytoplasmic dye.

Depending on the degree of relocalization you are looking for, you should keep in mind that most fixable surface stains will not stain the membrane itself, but structures on the membrane. So, given that your GFP moiety, when localized to the membrane, faces into the cytoplasm, there might be a considerable offset from your surface stain, even above the diffraction limit (assuming that you are performing diffraction-limited imaging). The closest you will be able to get is using a fixable lipophilic dye, such as CM-DiI, though that will also stain intracellular membranes as it is internalized. If you don't mind the offset factor, your best bet for surface staining is either using an antibody for an abundant surface protein (depending on your cell type, e.g. CD81), or a fluorophore-conjugated lectin like wheat germ agglutinin. You can also perform these stains after fixation (but before permeabilization), but keep in mind that fixation does lightly permeabilize the membrane, whereas a live cell stain on ice will be strictly on the surface. I guess you could theoretically also perform the CM-DiI stain immediately before fixation, though I have not tried that and don't know if it would still diffuse through the cell membrane in living cells.

I would also recommend a brief permeabilization step immediately after fixation (e.g. PBS/0.2% Triton X-100 for 10 min) to prevent post-fixation blebbing, which can happen to varying degrees depending on the cell type and even cellular PIP2 levels (see doi:10.1016/j.fob.2014.02.003), and which would affect your ability to accurately localize cell subtructures. I have battled post-fixation blebbing for a couple of years and now swear by the post-fixation perm step. If you are using CM-DiI (or any lipophilic dye), do not permeabilize with Triton as it will wash out, use digitonin instead.

Best regards,
Mel



On 3/2/2015 6:30 AM, Valeria Berno wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear all,
>
> One of the facility user is trying to quantify Inositol phosphates (PIP) in the plasma membrane of our cells. We manage to visualize it transfecting the cells with a recombinant GFP.
>
> The idea is that the GFP-PIP should migrate from membrane to cytoplasm with specific treatment. We counterstained the cells with a cytoplasmatic marker.
>
> We decided to quantify this with a line scan method  (drawing a line through the cells) and measure the ratio between intensity in the membrane and intensity in the cytoplasm. This is a very common method in different paper but I couldn´t find more specifics.
>
>
> My questions are:
> -how can I determine where is my membrane a priori only looking at the cytoplasmatic staining?.
> First of all the cytoplasmatic staining doesn´t drop in a specific postion but gradually decrease where I can clearly see the peak of the membrane.
>
> Assuming the membrane should start where the peak of the cytoplasm staining start to decrease...then should I assume that the membrane is always a specific width or should I calculate the GFP intensity up to when I reach background level for the green?
>
> I know that the best should be a membrane staining as a reference but could you suggest a good one for fixed cells?
>
> Thanks in advance for your support
>
> Valeria
>
>
> Valeria Berno, PhD
> Imaging Facility
> ______
>
> Istituto Nazionale di Genetica Molecolare Istutito Nazionale Genetica
> Molecolare "Romeo ed Enrica Invernizzi"
> Via F.Sforza 35 - 20122 - Milano (MI) - Italy Tel Off.: +39 02.00660
> 219 Tel Lab.:+39 02.00660 338
> E-Mail: [hidden email]
> Web site: http://www.ingm.org
>
> SOSTIENI LA RICERCA: devolvi il 5 per mille delle imposte alla FONDAZIONE INGM Istituto Nazionale Genetica Molecolare di Milano. Devi solo inserire il codice fiscale della Fondazione - 04175700964 - in tutti i modelli CUD, 730 e UNICO nella sezione relativa al finanziamento per la RICERCA SANITARIA. Grazie 1000...al 5 per mille!
> Please consider the environment before printing this mail note
>
> DISCLAIMER :
> Questa e-mail potrebbe contenere informazioni privilegiate o confidenziali. Se pensa di aver ricevuto questa e-mail per errore, è pregato di contattare il mittente rispondendo a questa stessa dopodichè è pregato di cancellarla immediatamente.
>
> This e-mail may contain confidential or privileged information. If you think you have received this e-mail in error, please advise the sender by reply e-mail and then delete this e-mail immediately.
>

--
Menelaos Symeonides
University of Vermont
Cell & Molecular Biology Graduate Program Department of Microbiology and Molecular Genetics
318 Stafford Hall
95 Carrigan Dr
Burlington, VT 05405
[hidden email]
Phone: [phone]