Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi all,
but also keep in mind that 30% variation in sensitivity between any two specimen of exactly the same type of PMT is common.
Confocaling is NOT a quantitative thing (yet).
Kees
kees jalink
Div. of Cell Biology, The Netherlands Cancer Institute
Plesmanlaan 121, 1066CX Amsterdam
Phone:31-20-512 1933, Fax:31-20-512 1944, [log in to unmask]
home address:
Paulus Potterlaan 5, 2102CC Heemstede, The Netherlands
Phone: 31-23-547 6047, [log in to unmask]
> -----Original Message-----
> From: Automatic digest processor
> [mailto:[log in to unmask]]
> Sent: Tuesday, May 13, 2003 6:06 AM
> To: Recipients of CONFOCAL digests
> Subject: CONFOCAL Digest - 9 May 2003 to 12 May 2003 (#2003-102)
>
>
> There are 11 messages totalling 664 lines in this issue.
>
> Topics of the day:
>
> 1. Controls in immunolabelled cells (5)
> 2. Leica SP-2 PMT sensitivity (4)
> 3. Isotype matched or whole immunoglobulin controls in
> immunolabelled cells
> 4. <No subject given>
>
> ----------------------------------------------------------------------
>
> Date: Mon, 12 May 2003 13:52:22 +0200
> From: Fredrik Swift <[log in to unmask]>
> Subject: Controls in immunolabelled cells
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello,
>
> I'm planning some immunolabelling experiments. I will use
> primary antibodies
> agains subunits of the Na/K-ATPase and the Na/Ca-exchanger in
> permeabilized
> rat cardiomyocytes and secondary antibodies coupled to an
> Alexa-fluorochrome
> (Mol. Probes). What would be appropriate controls in this type of
> experiment?
> - Without primary antibody?
> - Non permeabilized cells?
> - Any other good positive/negative controls?
>
> Thanks,
>
> Fredrik
>
> ------------------------------
>
> Date: Mon, 12 May 2003 14:26:31 +0200
> From: marco sassoe <[log in to unmask]>
> Subject: Re: Controls in immunolabelled cells
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Fredrik,
>
> I would incubate some sections with just one
> primary ab followed by both secondary abs.
>
> Best regards,
> Marco
>
>
>
> ----- Original Message -----
> From: Fredrik Swift <[log in to unmask]>
> Date: Monday, May 12, 2003 1:52 pm
> Subject: Controls in immunolabelled cells
>
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Hello,
> >
> > I'm planning some immunolabelling experiments. I will use primary
> > antibodiesagains subunits of the Na/K-ATPase and the Na/Ca-
> > exchanger in permeabilized
> > rat cardiomyocytes and secondary antibodies coupled to an Alexa-
> > fluorochrome(Mol. Probes). What would be appropriate controls in
> > this type of
> > experiment?
> > - Without primary antibody?
> > - Non permeabilized cells?
> > - Any other good positive/negative controls?
> >
> > Thanks,
> >
> > Fredrik
> >
>
> ------------------------------
>
> Date: Mon, 12 May 2003 14:47:50 +0100
> From: francesca magnani <[log in to unmask]>
> Subject: Re: Controls in immunolabelled cells
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Fredrik,
>
> for the first time I would set up the following controls:
> -no antibodies (for autofluorescence)
> -no primary antibodies, incubate only with secondary abs (to
> check whether
> the secondary abs bind aspecifically your sample)
> -I would also verify that the secondary ab does not
> aspecifically bind the
> other primary:
> i.e. if you have
> anti-A (raised in mouse)
> anti-B (raised in rabbit)
> anti-mouse
> anti-rabbit
>
> set up one slide incubating with anti-A (mo)+anti-rabbit
> and another one for anti-B (rb)+anti-mouse.
>
> Good luck,
> Francesca
>
>
>
>
> >----- Original Message -----
> >From: Fredrik Swift <[log in to unmask]>
> >Date: Monday, May 12, 2003 1:52 pm
> >Subject: Controls in immunolabelled cells
> >
> > > Search the CONFOCAL archive at
> > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > Hello,
> > >
> > > I'm planning some immunolabelling experiments. I will use primary
> > > antibodiesagains subunits of the Na/K-ATPase and the Na/Ca-
> > > exchanger in permeabilized
> > > rat cardiomyocytes and secondary antibodies coupled to an Alexa-
> > > fluorochrome(Mol. Probes). What would be appropriate controls in
> > > this type of
> > > experiment?
> > > - Without primary antibody?
> > > - Non permeabilized cells?
> > > - Any other good positive/negative controls?
> > >
> > > Thanks,
> > >
> > > Fredrik
> > >
>
> --------------------------------------------------------------
>
> Francesca Magnani
> Biochemistry Department
> Trinity College
> Dublin 2
> Ireland
>
> phone: 00353 1 6082445
> fax: 00353 1 6772400
> [log in to unmask]
>
> ------------------------------
>
> Date: Mon, 12 May 2003 09:01:52 -0500
> From: Karl Garsha <[log in to unmask]>
> Subject: Re: Controls in immunolabelled cells
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello Fredrik,
> The following link will take you to a PDF file of a short article
> discussing controls for immunolabeling (Microscopy Today,
> November 2001).
>
> http://www.itg.uiuc.edu/people/garsha/documents/Immunolabeling.pdf
>
> Cheers,
> Karl G.
>
> Fredrik Swift wrote:
>
> >Search the CONFOCAL archive at
> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> >Hello,
> >
> >I'm planning some immunolabelling experiments. I will use
> primary antibodies
> >agains subunits of the Na/K-ATPase and the Na/Ca-exchanger
> in permeabilized
> >rat cardiomyocytes and secondary antibodies coupled to an
> Alexa-fluorochrome
> >(Mol. Probes). What would be appropriate controls in this type of
> >experiment?
> >- Without primary antibody?
> >- Non permeabilized cells?
> >- Any other good positive/negative controls?
> >
> >Thanks,
> >
> >Fredrik
> >
> >
>
> ------------------------------
>
> Date: Mon, 12 May 2003 10:11:04 -0400
> From: "Perez, Dr. Guillermo" <[log in to unmask]>
> Subject: Re: Controls in immunolabelled cells
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
>
> Dear Frederik,
>
> We use NCX antibodies in canine cardiac myocytes.
> You need blocking peptide controls (pre-absorbed primary),
> secondary ab alone controls(no primary),
> background fluorescence controls (no secondary).
>
> I would not try double labeling at first, as you will increase your
> combinations to an out of control number of slides. This is important
> because you have to run your slides all at the same time same
> conditions, and of course when viewing them, same recording conditions
> as well (i.e. PMT, laser intensity, confocal aperture, etc).
> You need to
> find out your working dilutions also (for both primary and secondary
> abs) which complicates your combinations a bit more.
>
> Yours, Guillermo
>
>
>
> Guillermo J. Perez , Ph.D.
> Research Scientist I=20
> Masonic Medical Research Laboratory
> 2150 Bleecker Street
> Utica, NY 13510-1787
> (315) 735-2217 ext. 55 or 13
> =20
>
> -----Original Message-----
> From: Fredrik Swift [mailto:[log in to unmask]]=20
> Sent: Monday, May 12, 2003 7:52 AM
> To: [log in to unmask]
> Subject: Controls in immunolabelled cells
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
>
> Hello,
>
> I'm planning some immunolabelling experiments. I will use primary
> antibodies
> agains subunits of the Na/K-ATPase and the Na/Ca-exchanger in
> permeabilized
> rat cardiomyocytes and secondary antibodies coupled to an
> Alexa-fluorochrome
> (Mol. Probes). What would be appropriate controls in this type of
> experiment?
> - Without primary antibody?
> - Non permeabilized cells?
> - Any other good positive/negative controls?
>
> Thanks,
>
> Fredrik
>
> ------------------------------
>
> Date: Mon, 12 May 2003 09:13:41 -0500
> From: Karl Garsha <[log in to unmask]>
> Subject: Leica SP-2 PMT sensitivity
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Greetings,
> Has anybody determined a benchmark for evaluating PMT
> sensitivity on the
> SP-2 platform? I've recently discovered that our PMT 1 is showing
> roughly 30% of the sensitivity of PMT 3 and PMT 2 is showing
> 65% of the
> sensitivity of PMT 3 (at either end of the spectrum). This
> is a little
> alarming and I would like to determine whether PMT 3 is in fact
> performing well or if the sensitivity is down for it as well.
> Thanks in
> advance for any suggestions.
> Cheers,
> Karl G.
>
> ------------------------------
>
> Date: Mon, 12 May 2003 09:46:12 -0600
> From: Gayle Callis <[log in to unmask]>
> Subject: Isotype matched or whole immunoglobulin controls in
> immunolabelled cells
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear all,
>
> Sorry this is so long -
>
> Use permeabilized cells and substitute primary host
> immunoglobulin at the
> same working concentration as primary antibody. Example: if
> primary is
> mouse anti "subunits of the Na/K-ATPase", mouse IgG2a indicating
> immunoglobulin subclass of mouse host, then the negative
> control is Mouse
> IgG2a. Whole mouse IgG can be used since it is a mixture of
> all subclasses
> or isotypes. This will test if host immunglobulin portion causes
> nonspecific staining and this will not be tested IF you do just a PBS
> control. Some people will use primary antibody of irrelevant
> specificity
> in place of whole or isotype matched Ig's.
>
> Caveat: We did not do a proper isotype matched control once,
> and reviewers
> asked for repeat of immunostaining - a harsh lesson!
>
> PBS or diluent subtituted for a primary antibody is fine for checking
> nonspecific binding of secondary antibody to cells. We refer
> to PBS as a
> null control. I really liked "no antibodies for checking
> autofluorescence"
> - good point!
>
> To borrow from Francesca's tidy setup a bit, we would set up staining
> protocol:
> Permeabilized cells with:
>
> 1. Mouse IgG negative
> Goat antimouse F(ab') frag of whole IgG, adsorbed to rat
>
> 2. Mouse anti A
> Goat antimouse (F(ab')2 frag of whole IgG, adsorbed to rat
>
> 3. Rabbit IgG negative control
> Donkey anti Rabbit F(ab')2 fragment of IgG, adsorbed to rat
>
> 4. Rabbit anti B
> Donkey anti Rabbit F(ab')2 fragment of IgG, adsorbed to rat
>
> We use F(ab')2 fragments of IgG to prevent cross reaction to
> fc receptors
> on cells, particularly important in closely related species -
> rat and mouse
>
> In place of "set up one slide incubating with anti-A (mo)+anti-rabbit
> >and another one for anti-B (rb)+anti-mouse.", you can
> substitute Anti A
> antibody with mouse IgG, and rabbit IgG for anti-B, to test
> if secondary
> antibodies are cross reacting to primary antibody host
> immunoglobulins.
>
> It is a good idea for planned double staining, to do single
> staining first
> to determine nonspecific binding of Mouse IgG with its
> secondary and same
> for rabbit IgG with its secondary, make necessary
> adjustments, then combine
> for double staining.
>
> Gayle Callis
> Research Histopathology Supervisor
> Veterinary Molecular Biology
> Montana State University
> Bozeman MT 59717-3610
> email: [log in to unmask]
>
>
> >Hi Fredrik,
> >
> >for the first time I would set up the following controls:
> >-no antibodies (for autofluorescence)
> >-no primary antibodies, incubate only with secondary abs (to
> check whether
> >the secondary abs bind aspecifically your sample)
> >-I would also verify that the secondary ab does not
> aspecifically bind the
> >other primary:
> >i.e. if you have
> >anti-A (raised in mouse)
> >anti-B (raised in rabbit)
> >anti-mouse
> >anti-rabbit
> >
> >set up one slide incubating with anti-A (mo)+anti-rabbit
> >and another one for anti-B (rb)+anti-mouse.
> >
> >Good luck,
> >Francesca
> >
>
> >>From: Fredrik Swift <[log in to unmask]>
> >>Date: Monday, May 12, 2003 1:52 pm
> >>Subject: Controls in immunolabelled cells
> >> > Hello,
> >> >
> >> > I'm planning some immunolabelling experiments. I will use primary
> >> > antibodiesagains subunits of the Na/K-ATPase and the Na/Ca-
> >> > exchanger in permeabilized
> >> > rat cardiomyocytes and secondary antibodies coupled to an Alexa-
> >> > fluorochrome(Mol. Probes). What would be appropriate controls in
> >> > this type of
> >> > experiment?
> >> > - Without primary antibody?
> >> > - Non permeabilized cells?
> >> > - Any other good positive/negative controls?
> >> >
> >> > Thanks,
> >> >
> >> > Fredrik
>
> Gayle Callis
> MT,HT,HTL(ASCP)
> Research Histopathology Supervisor
> Veterinary Molecular Biology - Marsh Lab
> Montana State University - Bozeman
> S. 19th and Lincoln St
> Bozeman MT 59717-3610
>
> 406 994-6367 (lab with voice mail)
> 406 994-4303 (FAX)
>
> email: [log in to unmask]
>
> ------------------------------
>
> Date: Tue, 13 May 2003 02:35:35 +1000
> From: Guy Cox <[log in to unmask]>
> Subject: Re: Leica SP-2 PMT sensitivity
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Quoting Karl Garsha <[log in to unmask]>:
>
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Greetings,
> > Has anybody determined a benchmark for evaluating PMT
> sensitivity on the
> > SP-2 platform? I've recently discovered that our PMT 1 is showing
> > roughly 30% of the sensitivity of PMT 3 and PMT 2 is
> showing 65% of the
> > sensitivity of PMT 3 (at either end of the spectrum). This
> is a little
> > alarming and I would like to determine whether PMT 3 is in fact
> > performing well or if the sensitivity is down for it as
> well. Thanks in
> > advance for any suggestions.
>
> Depending on your system they may well be different types of PMT -
> I think our system has 1 bi-alkali and the others multi-alkali. So
> check that first. Generally one would expect that more
> blue-sensitivity
> is desirable on PMT1 and more red on PMT3. The other thing
> to remember
> is that voltage response may vary and a more accurate control is
> obtainable signal to noise ratio.
>
> Guy
>
> --
> Associate Professor Guy Cox
> Electron Microscope Unit, F09
> University of Sydney NSW 2006
> +61 2 9351 3176
>
> ------------------------------
>
> Date: Mon, 12 May 2003 12:52:54 -0400
> From: Russell McConnell <[log in to unmask]>
> Subject: Re: Leica SP-2 PMT sensitivity
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> One more thing to keep in mind is that the scan head
> configuration also
> plays into it. Depending on where the PMT's are located, one of them
> receives the light 'directly' from the prism system, whereas the other
> PMT's are getting their light bounced off of the mirrors that control
> the spectral window for that first PMT (is usually PMT 2 I
> believe). Of
> course mirror losses should be fairly minimal, so this is probably not
> the only factor here. I do think it is pretty important however.
> -Russell
>
> Guy Cox wrote:
> >
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Quoting Karl Garsha <[log in to unmask]>:
> >
> > > Search the CONFOCAL archive at
> > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > > Greetings,
> > > Has anybody determined a benchmark for evaluating PMT
> sensitivity on the
> > > SP-2 platform? I've recently discovered that our PMT 1 is showing
> > > roughly 30% of the sensitivity of PMT 3 and PMT 2 is
> showing 65% of the
> > > sensitivity of PMT 3 (at either end of the spectrum).
> This is a little
> > > alarming and I would like to determine whether PMT 3 is in fact
> > > performing well or if the sensitivity is down for it as
> well. Thanks in
> > > advance for any suggestions.
> >
> > Depending on your system they may well be different types of PMT -
> > I think our system has 1 bi-alkali and the others multi-alkali. So
> > check that first. Generally one would expect that more
> blue-sensitivity
> > is desirable on PMT1 and more red on PMT3. The other thing
> to remember
> > is that voltage response may vary and a more accurate control is
> > obtainable signal to noise ratio.
> >
> > Guy
> >
> > --
> > Associate Professor Guy Cox
> > Electron Microscope Unit, F09
> > University of Sydney NSW 2006
> > +61 2 9351 3176
>
> --
> Russell McConnell
> Confocal Imaging Facility Manager
> Department of Neuroscience
> Tufts University
> 136 Harrison Ave.
> Boston, MA 02111
> Tel. (617) 636-3795
> http://www.neurosci.tufts.edu/Imaging
>
> ------------------------------
>
> Date: Tue, 13 May 2003 00:54:52 +0800
> From: Chen Yuan Dong <[log in to unmask]>
> Subject: <No subject given>
>
> This is a multi-part message in MIME format.
>
> ------=_NextPart_000_006C_01C318EA.436FA530
> Content-Type: text/plain;
> charset="big5"
> Content-Transfer-Encoding: quoted-printable
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
>
> Hi Everyone,
> I have been looking for cover slips that are index matched
> to water. I know that you could get them from Olympus in
> the past. If anyone has any information on where (and the
> cost) of these cover slips, it will be greatly appreciated if you
> can let me know.
>
> Sincerely,
>
> Chen-Yuan
> _______________________________________
>
> Chen-Yuan Dong, Ph.D.
> Assistant Professor
> Microscopic Biophysics Laboratory
> Department of Physics
> National Taiwan University
> Taipei 106, Taiwan
> Republic of China
> Tel: 8862-2-3366-5155 (Office, Room 530)=20
> 8862-2-3366-5196 (Lab, Room 830)
> Fax: 886-2-2363-9984
> E-mail: [log in to unmask]
>
>
> ------=_NextPart_000_006C_01C318EA.436FA530
> Content-Type: text/html;
> charset="big5"
> Content-Transfer-Encoding: quoted-printable
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=3Dconfocal
> <!DOCTYPE HTML PUBLIC "-//W3C//DTD HTML 4.0 Transitional//EN">
> <HTML><HEAD>
> <META http-equiv=3DContent-Type content=3D"text/html; charset=3Dbig5">
> <META content=3D"MSHTML 6.00.2600.0" name=3DGENERATOR>
> <STYLE></STYLE>
> </HEAD>
> <BODY bgColor=3D#ffffff>
> <DIV><FONT size=3D2>Hi Everyone,</FONT></DIV>
> <DIV><FONT size=3D2> I have been looking for cover =
> slips that=20
> are index matched</FONT></DIV>
> <DIV><FONT size=3D2>to water. I know that you could get them from =
> Olympus=20
> in</FONT></DIV>
> <DIV><FONT size=3D2>the past. If anyone has any information on where =
> (and=20
> the</FONT></DIV>
> <DIV><FONT size=3D2>cost) of these cover slips, it will be greatly =
> appreciated if=20
> you</FONT></DIV>
> <DIV><FONT size=3D2>can let me know.</FONT></DIV>
> <DIV> </DIV>
> <DIV><FONT size=3D2>Sincerely,</FONT></DIV>
> <DIV> </DIV>
> <DIV><FONT=20
> size=3D2>Chen-Yuan<BR>_______________________________________<
> /FONT></DIV=
> >
> <DIV> </DIV>
> <DIV><FONT size=3D2>Chen-Yuan Dong, Ph.D.<BR>Assistant =
> Professor<BR>Microscopic=20
> Biophysics Laboratory<BR>Department of Physics<BR>National Taiwan=20
> University<BR>Taipei 106, Taiwan<BR>Republic of China<BR>Tel: =20
> 8862-2-3366-5155 (Office, Room 530)=20
> <BR>
> 8862-2-3366-5196 (Lab, =
> Room=20
> 830)<BR>Fax: 886-2-2363-9984<BR>E-mail: <A=20
> href=3D"mailto:[log in to unmask]">[log in to unmask]<
> /A></FONT><=
> /DIV>
> <DIV><FONT size=3D2></FONT> </DIV></BODY></HTML>
>
> ------=_NextPart_000_006C_01C318EA.436FA530--
>
> ------------------------------
>
> Date: Tue, 13 May 2003 08:42:47 +1000
> From: Rosemary White <[log in to unmask]>
> Subject: Re: Leica SP-2 PMT sensitivity
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Karl,
>
> Guy is correct here. I've checked our 4 PMTs against bluish
> through deep
> red fluorescence, and PMT1 is definitely most sensitive in
> the blue, PMT2
> in green-yellow, PMT3 in orange-red and PMT4 in red through long red
>
> Another quirk to be aware of is that the dichroic filters let through
> different % of laser light. So the intensity of the 488 line coming
> through RSP500, DD488/543 and TD488/543/633 is different for each.
>
> cheers,
> Rosemary
>
> >Search the CONFOCAL archive at
> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> >Quoting Karl Garsha <[log in to unmask]>:
> >
> >> Search the CONFOCAL archive at
> >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>
> >> Greetings,
> >> Has anybody determined a benchmark for evaluating PMT
> sensitivity on the
> >> SP-2 platform? I've recently discovered that our PMT 1 is showing
> >> roughly 30% of the sensitivity of PMT 3 and PMT 2 is
> showing 65% of the
> >> sensitivity of PMT 3 (at either end of the spectrum).
> This is a little
> >> alarming and I would like to determine whether PMT 3 is in fact
> >> performing well or if the sensitivity is down for it as
> well. Thanks in
> >> advance for any suggestions.
> >
> >Depending on your system they may well be different types of PMT -
> >I think our system has 1 bi-alkali and the others multi-alkali. So
> >check that first. Generally one would expect that more
> blue-sensitivity
> >is desirable on PMT1 and more red on PMT3. The other thing
> to remember
> >is that voltage response may vary and a more accurate control is
> >obtainable signal to noise ratio.
> >
> > Guy
> >
> >--
> >Associate Professor Guy Cox
> >Electron Microscope Unit, F09
> >University of Sydney NSW 2006
> >+61 2 9351 3176
>
>
> Rosemary White
> Microscopy Centre
> CSIRO Plant Industry, Canberra
> phone 6246 5475 or 0402 835 973
> email [log in to unmask]
>
> ------------------------------
>
> End of CONFOCAL Digest - 9 May 2003 to 12 May 2003 (#2003-102)
> **************************************************************
>
|
