[Histonet] Re: Histonet Digest, Vol 111, Issue 5

Tue Feb 5 13:42:52 CST 2013

Hi Brian,

I am wondering three possibilities: 1. the PFA that you use, powder form,
is rather old. ( however, you said that you have tried other powders and
concoctions that didn't work as well!)... perhaps, you could try using
Polysciences Formaldehyde. The sealed vials come as 16% stock that you can
open and dilute immediately.

2. The procedure:  For some reason, the fixative isn't reaching the brain
well enough. The question to you is, do you see clear, whitish coloured
brain after you dissect or would you see it reddish or pinkish in colour.
The reason could be a. the heart stopped beating very early in your
procedure. Perhaps, slightly lower the dose or Avertin/anaesthetic. (I have
had this issue previously, but it almost always works if the brain sits in
the fixative unless it is never fixed! Or, the needle is not positioned
correctly in the left ventricle, and fixative did not reach circulation.

3.Fixation itself:  I Have had issues with the dendrites looking bloated
and nobby with swiss cheese morphology before with only PFA fixation.
These, however, were slice cultures, so different from whole brain
fixations. They apparently needed 4% sucrose in the fixative to take care
of the   However,  I would definitely check that. I would suggest adding 4%
sucrose to the fixative as well and see if it improves the morphology.

Please let us know what worked for you when you solve the issue!!!  Thanks
and Best,

Pow

Message: 4

> Date: Mon, 04 Feb 2013 15:38:51 -0800
> From: "Brian W. Jones" <bwjones <@t> uw.edu>
> Subject: [Histonet] extensive troubleshooting of IF on
>         fixed-then-frozen       mouse brain
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <5110468B.1050607 <@t> uw.edu>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> Hi All,
>
> I've used Histonet many times to answer my IF/IHC questions, but this
> time I'm stumped. We've been using a transcardial perfusion
> fixation/immunofluoresence protocol for years and it has always worked.
> It has worked so reliably that we would often make last-minute
> changes---e.g., post-fix overnight instead of 4 hours---if something
> else came up and it would still turn out fine.
>
> Well, obviously things aren't working now or I wouldn't be pleading for
> help!
>
> THE PROBLEMS:
> Mouse brain tissue has large holes (Swiss cheese look), lacks fine
> structures such as axons or dendrites (looks mushy), some of the brain
> regions easily separate away from others (e.g., the hippocampus
> separates from the thalamus), and random areas of the section are not in
> the same focal plane as the surrounding tissue. (I tried to post images
> as instructed on the web site, but I can't figure that out. Please
> advise as I think the images would show better than I can describe.)
>
> THE PROTOCOL:
> Here is a detailed description of the protocol and variations I have
> tried while troubleshooting. Note that I have not tried every
> combination of every variation, and certainly I am careful to only
> change one thing at a time so I can make meaningful comparisons.
>
> 1) Euthanize 3 - 5-month old mouse with Beuthansia D. Wait until no
> longer responsive to front paw pinch and restrain animal on dissecting
> board. Make incision to expose thoracic cavity and lift ribcage out of
> the way. Heart is still beating strongly at this point.
> 2) Grasp heart with blunt forceps and a) make small incision in left
> ventricle and insert 20-g cannula, or b) insert 20-g or 25-g needle into
> left ventricle.
> 3) Using a peristaltic pump, perfuse PBS and immediately cut right
> atrium for outflow. Have tried PBS at either 4 C or 35 C. Have made up
> PBS fresh and checked pH. Have replaced tubing and used a different pump.
> 4) Set flow rate to a) 1.5 mL/min, b) 3 mL/min, c) 6 mL/min, or d) 10
> mL/min. (Do not have a way to measure pressure.)
> 5) Perfuse PBS: a) 3 mL only, b) until liver blanches (never really
> blanches at low flow rates), or c) 15 mL.
> 6) PBS is either a) PBS alone, or b) PBS with heparin.
> 7) Perfuse with 50 mL of fixative: 4% PFA in PBS or PLP (Nakane)
> fixative. Note: I have tried PFA from Sigma (two different lots) or EMS
> (granules or 16%). Fixative is perfused at same rate and temp as PBS.
> 8) For 8% PFA stock: PFA is usually dissolved in water but have tried
> directly in PBS. Dissolved by bringing solution to 55 C then adding PFA
> while stirring. A few drops of 1-4 M NaOH are added until solution
> clears (a few undissolved particles remain). Solution is filtered
> through Whatman paper or 0.45 um membrane. Always made fresh the day of
> the perfusion.
> 9) Animal's lower extremities begin to move shortly after introduction
> of fixative. Rarely observe perfusate exiting the nose/mouth.
> 10) Post-fix: after perfusion, brain is dissected from skull using
> scissors and forceps and placed in 4 C fixative for 4-7 hours or overnight.
> 11) Brain is cryoprotected in sucrose-PBS: a) 10% sucrose until brain
> sinks, then 20%, then 30%; or b) 30% sucrose until brain sinks; c) three
> changes of 25% sucrose over three days total, or d) some other variation
> that ultimately ends in a brain sunk in at least 25% sucrose.
> 12) Brain is placed in 4 C Tissue-Tek OCT and frozen a) on powdered dry
> ice, or b) liquid nitrogen and then on dry ice while freezing other brains.
> 13) Brain is stored at -80 C or taken directly to cryostat. Have used
> two different cryostats.
> 14) Brains are allowed to equilibrate to cryostat temp, usually -17 C.
> 15) 20 um sections with new knife are a) placed thawed directly onto
> slide or b) placed in PBS and floated onto slide. Note: everything
> "feels" right on the cryostat.
> 16) Slides: a) VWR Superfrost, b) Fisher Superfrost Plus, or c)
> Superfrost Plus Gold.
> 17) Sections on slides are a) left to air dry for 30+ minutes, b) stored
> -80 C, or c) placed immediately in TBS-T.
> 18) Antibody procedures are done in 1% BSA in TBS-T; washes are with
> TBS-T. (Have tried using another lab's TBS-T as well.)
> 19) Slides are mounted with Prolong Gold + DAPI --- and by this time
> I've gone through several bottles of it, so it's never old!
>
> As mentioned above, in the past it seemed that any variation to the
> protocol would still work. Now, it seems that I can't make it work no
> matter what I change. I was convinced that it was a fixative problem,
> but as noted I've tried multiple sources/preparations of PFA. As my
> colleague put it: "You've tested all the scientific and all the
> superstitious factors."
>
> Thank you for any help you can provide!
>
> --
> Brian Jones, Ph.D
>
>
> Message: 8
> Date: Tue, 5 Feb 2013 08:08:16 +0000
> From: Jonathan Cremer <Jonathan.Cremer <@t> med.kuleuven.be>
> Subject: RE: [Histonet] extensive troubleshooting of IF on
>         fixed-then-frozen       mouse brain
> To: "Brian W. Jones" <bwjones <@t> uw.edu>,
>         "histonet <@t> lists.utsouthwestern.edu"
>         <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
>         <
> C35C2709C22FC2458D6A918FACC338A30FA7A9C6 <@t> ICTS-S-MBX5.luna.kuleuven.be>
>
> Content-Type: text/plain; charset="iso-8859-1"
>
> You're not making your formaldehyde solution properly, that might be the
> first of your issues.
> Paraformaldehyde is the polymer of formaldehyde, and has little to no
> fixative properties. It doesn't dissolve in water either. To make a proper
> fixative from paraformaldehyde, you have to hydrolyse it in a basic
> solution.
>
> To make 4% formaldehyde in PBS, resuspend 40 g of paraformaldehyde in
> about 700 ml of water and add one or two drops of 2-3 M NaOH. Leave it
> stirring for 5-10 minutes, add one drop of NaOH if the solution hasn't
> cleared up entirely. (You can warm up the liquid to speed to process, but
> don't go above 60 °C or the formaldehyde will start to decompose.) Don't
> overdo it with the NaOH, because you have to bring down the pH again with
> HCl, and the more you add, the more the salinity of the final solution will
> go up.
> When you have a clear solution, add either 100 ml of 10x PBS, or add the
> dry powders for 1 l of PBS. Stir to dissolve/mix, then bring the pH to 7,4
> (or whichever desired) with 1 M HCl. Finally, add water to a total volume
> of 1 l.
>
> To my view, the PBS is there mostly to buffer the formic acid from
> formaldehyde decomposition. You could make up in water or saline only, but
> keep an eye on the pH.
>
> Also, when diluting concentrated formaldehyde (formalin and formal are
> just old commercial names for 37-41% of formaldehyde in water, with 10%
> methanol), use PBS for immediate use (the small polymers are then
> hydrolized rapidly) or dilute in water and leave 48 hours before use (slow
> depolymerization).
> ---
> Jonathan Cremer
> Laboratory Technician
> TARGID - KU Leuven
>
> Gasthuisberg CDG; Labo Experimentele Immunologie; Herestraat 49 bus 811;
> 3000 Leuven; Belgium
>
> ________________________________________
> Van: histonet-bounces <@t> lists.utsouthwestern.edu [
> histonet-bounces <@t> lists.utsouthwestern.edu] namens Brian W. Jones [
> bwjones <@t> uw.edu]
> Verzonden: dinsdag 5 februari 2013 0:38
> To: histonet <@t> lists.utsouthwestern.edu
> Onderwerp: [Histonet] extensive troubleshooting of IF on fixed-then-frozen
>      mouse brain
>
> Hi All,
>
> I've used Histonet many times to answer my IF/IHC questions, but this
> time I'm stumped. We've been using a transcardial perfusion
> fixation/immunofluoresence protocol for years and it has always worked.
> It has worked so reliably that we would often make last-minute
> changes---e.g., post-fix overnight instead of 4 hours---if something
> else came up and it would still turn out fine.
>
> Well, obviously things aren't working now or I wouldn't be pleading for
> help!
>
> THE PROBLEMS:
> Mouse brain tissue has large holes (Swiss cheese look), lacks fine
> structures such as axons or dendrites (looks mushy), some of the brain
> regions easily separate away from others (e.g., the hippocampus
> separates from the thalamus), and random areas of the section are not in
> the same focal plane as the surrounding tissue. (I tried to post images
> as instructed on the web site, but I can't figure that out. Please
> advise as I think the images would show better than I can describe.)
>
> THE PROTOCOL:
> Here is a detailed description of the protocol and variations I have
> tried while troubleshooting. Note that I have not tried every
> combination of every variation, and certainly I am careful to only
> change one thing at a time so I can make meaningful comparisons.
>
> 1) Euthanize 3 - 5-month old mouse with Beuthansia D. Wait until no
> longer responsive to front paw pinch and restrain animal on dissecting
> board. Make incision to expose thoracic cavity and lift ribcage out of
> the way. Heart is still beating strongly at this point.
> 2) Grasp heart with blunt forceps and a) make small incision in left
> ventricle and insert 20-g cannula, or b) insert 20-g or 25-g needle into
> left ventricle.
> 3) Using a peristaltic pump, perfuse PBS and immediately cut right
> atrium for outflow. Have tried PBS at either 4 C or 35 C. Have made up
> PBS fresh and checked pH. Have replaced tubing and used a different pump.
> 4) Set flow rate to a) 1.5 mL/min, b) 3 mL/min, c) 6 mL/min, or d) 10
> mL/min. (Do not have a way to measure pressure.)
> 5) Perfuse PBS: a) 3 mL only, b) until liver blanches (never really
> blanches at low flow rates), or c) 15 mL.
> 6) PBS is either a) PBS alone, or b) PBS with heparin.
> 7) Perfuse with 50 mL of fixative: 4% PFA in PBS or PLP (Nakane)
> fixative. Note: I have tried PFA from Sigma (two different lots) or EMS
> (granules or 16%). Fixative is perfused at same rate and temp as PBS.
> 8) For 8% PFA stock: PFA is usually dissolved in water but have tried
> directly in PBS. Dissolved by bringing solution to 55 C then adding PFA
> while stirring. A few drops of 1-4 M NaOH are added until solution
> clears (a few undissolved particles remain). Solution is filtered
> through Whatman paper or 0.45 um membrane. Always made fresh the day of
> the perfusion.
> 9) Animal's lower extremities begin to move shortly after introduction
> of fixative. Rarely observe perfusate exiting the nose/mouth.
> 10) Post-fix: after perfusion, brain is dissected from skull using
> scissors and forceps and placed in 4 C fixative for 4-7 hours or overnight.
> 11) Brain is cryoprotected in sucrose-PBS: a) 10% sucrose until brain
> sinks, then 20%, then 30%; or b) 30% sucrose until brain sinks; c) three
> changes of 25% sucrose over three days total, or d) some other variation
> that ultimately ends in a brain sunk in at least 25% sucrose.
> 12) Brain is placed in 4 C Tissue-Tek OCT and frozen a) on powdered dry
> ice, or b) liquid nitrogen and then on dry ice while freezing other brains.
> 13) Brain is stored at -80 C or taken directly to cryostat. Have used
> two different cryostats.
> 14) Brains are allowed to equilibrate to cryostat temp, usually -17 C.
> 15) 20 um sections with new knife are a) placed thawed directly onto
> slide or b) placed in PBS and floated onto slide. Note: everything
> "feels" right on the cryostat.
> 16) Slides: a) VWR Superfrost, b) Fisher Superfrost Plus, or c)
> Superfrost Plus Gold.
> 17) Sections on slides are a) left to air dry for 30+ minutes, b) stored
> -80 C, or c) placed immediately in TBS-T.
> 18) Antibody procedures are done in 1% BSA in TBS-T; washes are with
> TBS-T. (Have tried using another lab's TBS-T as well.)
> 19) Slides are mounted with Prolong Gold + DAPI --- and by this time
> I've gone through several bottles of it, so it's never old!
>
> As mentioned above, in the past it seemed that any variation to the
> protocol would still work. Now, it seems that I can't make it work no
> matter what I change. I was convinced that it was a fixative problem,
> but as noted I've tried multiple sources/preparations of PFA. As my
> colleague put it: "You've tested all the scientific and all the
> superstitious factors."
>
> Thank you for any help you can provide!
>
> --
> Brian Jones, Ph.D
>
>
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>